Lipid nanoparticle

Abstract

The purpose of the present invention is to provide a lipid nanoparticle that makes it possible to efficiently introduce a drug into a cell, and a constituent lipid of this lipid nanoparticle. Provided are: a compound represented by general formula (I), (II), or (III); a salt or stereoisomer thereof; and a lipid nanoparticle containing the same as a constituent lipid.

Description

lipid nanoparticles

[0001] The present invention relates to lipid nanoparticles that can efficiently deliver drugs into cells, and to the constituent lipids of the lipid nanoparticles.

[0002] Lipid nanoparticles (LNPs) are used as carriers for encapsulating lipid-soluble drugs or nucleic acids such as siRNA (small interfering RNA) or mRNA and delivering them to target cells. For example, lipid nanoparticles containing, as constituent lipids, pH-sensitive cationic lipids that are electrically neutral at physiological pH but change to cationic in a weakly acidic pH environment such as an endosome have been reported as lipid nanoparticles that serve as carriers for efficiently delivering nucleic acids such as siRNA into target cells.

[0003] The present applicants have disclosed lipid nanoparticles containing pH-sensitive cationic lipids that exhibit higher in vivo gene expression activity than when CL4F6 and CL15F6 are used as lipids (Patent Document 1). Furthermore, the present applicants have used retinoid conjugates in which constituent lipids are linked to a retinoid via a linker when forming siRNA-containing liposomes containing an ionizable cationic lipid (Patent Document 2). However, the lipids that can be used in lipid nanoparticles for drug delivery are limited, and the development of new lipids that can efficiently deliver drugs into cells has been desired.

[0004] International Publication No. 2022 / 071582 Special Publication No. 2015-523990

[0005] An object of the present invention is to provide lipid nanoparticles that can efficiently deliver drugs into cells, and the constituent lipids of the lipid nanoparticles.

[0006] The present inventors have discovered that lipid nanoparticles containing, as constituent lipids, pH-sensitive cationic lipids having a dioxane structure such as 1,3-dioxane or a dioxolane structure such as 1,3-dioxolane can efficiently deliver drugs into cells, and have completed the present invention.

[0007] That is, the present invention provides the following lipid nanoparticles and their constituent lipids: [1] General Formula I: General formula II: Or, general formula III: wherein L is independently H, straight-chain C １－１０ Alkyl or branched C ３－１０ Alkyl, or and A １ is C １－６ alkylene or phenylene; ２ is —OC(═O)—, —OC(═O)—O—, or —OC(═O)—NH—, or is absent; A ３ is C １－６ alkylene or absent, A ４ is a 4- to 15-membered nitrogen-containing heterocyclyl, wherein the nitrogen-containing heterocyclyl is unsubstituted or １－４ substituted with at least one selected from the group consisting of alkyl, hydroxy, and oxo; or A ４ teeth, where B １ and B ２ are each independently １－４ Alkyl, hydroxy C １－４ Alkyl, C １－４ Alkoxy C １－４ alkyl, or 4- to 6-membered heterocyclyl, wherein said 4- to 6-membered heterocyclyl is unsubstituted or １－４ is substituted with at least one selected from the group consisting of alkyl, hydroxy, and oxo; １ and M ２ are independently —O—, —O—CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -CH ２-, -O-C(O)-C-C-C(O)-, -O-C(O)-CH ２ -CH ２ -C(O)-O-, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -, -OC(O)-CH ２ -CH ２ -C(O)-NH-, -O-CH ２ -CH ２ -CH ２ -O-C(O)-, -C-C(O)-O-, -CH ２ -C(O)-O-CH ２ -, -C-CH ２ -O-C(O)-, -S-S-CH ２ - or -S-CH ２ -C-C-S-S-CH ２ - and R １ and R ２ are independently linear C １－３０ alkyl, branched C ３－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, sterol groups, and in general formulas I and III, at least one L is a compound represented by the formula (I), a salt thereof or a stereoisomer thereof.

[0008] [2] The compound is represented by general formula I, wherein one L is H and the other L is [3] The compound according to [1], a salt thereof, or a stereoisomer thereof, wherein one L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ The compound, salt or stereoisomer thereof according to [1] or [2], wherein

[0009] [4] One L is H and the other L is (In the formula, Rａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ The compound, salt or stereoisomer thereof according to any one of [1] to [3], wherein

[0010] [5] M １ and M ２ [6] The compound, salt or stereoisomer thereof according to any one of [1] to [4], wherein M １ and M ２ is -O-, -O-CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -or-CH ２ -CH ２ [7] The compound according to [5], a salt thereof, or a stereoisomer thereof, wherein R is —O—C(O)—. １ and R ２ are independently linear C １－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, and R ４ and R ５ are independently linear C １―１６ The compound, salt or stereoisomer thereof according to any one of [1] to [6], wherein R is alkyl.

[0011] [8] One L is H and the other L is and R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl, provided that R ａ and R ｂ The total number of carbon atoms in R is 4 or less, ｃ is a straight chain C ２－６[9] The compound according to any one of [1] to [7], a salt thereof, or a stereoisomer thereof, wherein R is alkylene. １ and R ２ is independently and R ４ and R ５ are independently linear C １―１１ alkyl, where R ４ and R ５ The compound, salt, or stereoisomer thereof according to any one of [1] to [7], wherein the total number of carbon atoms in R is 10 to 22. １ and R ２ is independently and R ４ and R ５ are independently linear C ５―１１ alkyl, where R ４ and R ５ The compound, salt or stereoisomer thereof according to any one of [1] to [7], wherein the difference in the number of carbon atoms between

[0012]

[11] The compound is represented by the general formula II, wherein L is a linear C １－５ alkyl, M １ and M ２ is -O-C(O)-, and R １ and R ２ are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６

[12] The compound according to [1], a salt thereof, or a stereoisomer thereof, wherein the compound is represented by general formula III, wherein one L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ alkylene), and M １ and M ２ is -O-C(O)-, and R １ and R ２are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６ The compound, salt or stereoisomer thereof according to [1], wherein R is alkyl.

[0013]

[13] The compound according to [1], a salt thereof, or a stereoisomer thereof, wherein the compound is any one of the following:

[0014]

[14] Lipid nanoparticles containing the compound according to any one of [1] to

[13] , or a salt or stereoisomer thereof.

[15] The lipid nanoparticles according to

[14] , further containing a sterol and a polyalkylene glycol-modified lipid.

[16] The lipid nanoparticles according to

[14] or

[15] , containing a nucleic acid.

[17] The lipid nanoparticles according to

[16] , wherein the nucleic acid is mRNA or siRNA.

[18] A pharmaceutical composition comprising the lipid nanoparticles according to any one of

[14] to

[17] .

[19] The pharmaceutical composition according to

[18] , used for gene therapy.

[20] A method for expressing an exogenous gene, comprising administering the lipid nanoparticles according to any one of

[14] to

[17] , encapsulating an exogenous gene to be expressed in cells, to a subject animal (excluding humans), and expressing the exogenous gene in the cells of the subject animal.

[0015] The lipid nanoparticles according to the present invention can efficiently deliver drugs into cells, making them useful as gene delivery carriers for gene therapy.

[0016] Hereinafter, embodiments of the present invention will be specifically described.

[0017] The lipid nanoparticles according to the present invention are lipid nanoparticles containing a pH-sensitive cationic lipid (hereinafter sometimes referred to as the "pH-sensitive cationic lipid of the present invention") having a dioxane structure or dioxolane structure represented by the following general formula (I), (II), or (III): By including a pH-sensitive cationic lipid that is a compound represented by general formula (I), (II), or (III), or a salt or stereoisomer thereof as a constituent lipid of the lipid nanoparticles, the lipid nanoparticles according to the present invention can efficiently introduce drugs into cells.

[0018] General formula (I)

[0019] General formula (II)

[0020] General formula (III)

[0021] In the general formulas (I) to (III), L is independently H, linear C １－１０ Alkyl or branched C ３－１０ Alkyl, or and A １ is C １－６ alkylene or phenylene; ２ is —OC(═O)—, —OC(═O)—O—, or —OC(═O)—NH—, or is absent; A ３ is C １－６ alkylene or absent, A ４ is a 4- to 15-membered nitrogen-containing heterocyclyl, wherein the nitrogen-containing heterocyclyl is unsubstituted or １－４ substituted with at least one selected from the group consisting of alkyl, hydroxy, and oxo; or A ４ teeth, where B １ and B ２ are each independently １－４ Alkyl, hydroxy C １－４ Alkyl, C １－４ Alkoxy C １－４alkyl, or 4- to 6-membered heterocyclyl, wherein said 4- to 6-membered heterocyclyl is unsubstituted or １－４ It is substituted with at least one selected from the group consisting of alkyl, hydroxy and oxo.

[0022] In the general formulas (I) and (III), at least one of L is is.

[0023] When the compound is represented by general formula (I), one L is H and the other L is It is preferable that:

[0024] In one embodiment of general formulas (I) to (III), L is independently H, linear or branched C １－１０ Alkyl, (In the formula, R ａ and R ｂ are independently linear or branched C １－３ alkyl, and R ｃ is a linear or branched C １－６ alkylene).

[0025] When the compound is represented by general formula (I), more preferably, one L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ alkylene).

[0026] When the compound is represented by general formula (I), more preferably, one L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ alkylene).

[0027] In the general formulas (I) to (III), M １ and M ２are independently —O—, —O—CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -CH ２ -, -O-C(O)-C-C-C(O)-, -O-C(O)-CH ２ -CH ２ -C(O)-O-, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -, -OC(O)-CH ２ -CH ２ -C(O)-NH-, -O-CH ２ -CH ２ -CH ２ -O-C(O)-, -C-C(O)-O-, -CH ２ -C(O)-O-CH ２ -, -C-CH ２ -O-C(O)-, -S-S-CH ２ - or -S-CH ２ -C-C-S-S-CH ２ In one embodiment of the present invention, M １ and M ２ are identical.

[0028] In general formula (I), M １ and M ２ are preferably the same. １ and M ２ When are the same, M １ and M ２ is -O-, -O-CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH２ -, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -or-CH ２ -CH ２ -O-C(O)-.

[0029] In general formulas (I) to (III), R １ and R ２ are independently linear or branched C １－３０ alkyl, linear or branched C ２－３０ alkenyl, adamantyl, sterol groups, (In the formula, R ４ and R ５ are independently linear or branched C １―１６ In one embodiment of the present invention, R １ and R ２ are identical.

[0030] In another embodiment of general formulas (I) to (III), R １ and R ２ are independently linear C １－３０ alkyl, branched C ３－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, sterol groups, is.

[0031] In general formula (I), preferably, R １ and R ２ are independently linear C １－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, and R ４ and R ５ are independently linear C １―１６ It is alkyl.

[0032] In one embodiment of general formula (I), one L is H and the other L is and R ａ and R ｂare independently methyl, ethyl, n-propyl, or isopropyl, provided that R ａ and R ｂ The total number of carbon atoms in R is 4 or less, ｃ is a straight chain C ２－６ alkylene, where preferably R １ and R ２ is independently and R ４ and R ５ are independently linear C １―１１ alkyl, where R ４ and R ５ The total number of carbon atoms in R is preferably 10 to 22, and more preferably R １ and R ２ is independently and R ４ and R ５ are independently linear C ５―１１ alkyl, where R ４ and R ５ The difference in the number of carbon atoms is 0 to 3.

[0033] In one embodiment of general formula (II), L is a linear C １－５ alkyl, M １ and M ２ is -O-C(O)-, and R １ and R ２ are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６ It is alkyl.

[0034] In one embodiment of general formula (III), one L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ alkylene), and M １ and M ２is -O-C(O)-, and R １ and R ２ are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６ It is alkyl.

[0035] The pH-sensitive cationic lipid of the present invention is, for example, a pH-sensitive cationic lipid having a dioxane structure or a dioxolane structure, and having the following structure, a salt thereof, or a stereoisomer thereof:

[0036] Compound 1: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-hexyldecanoate)

[0037] Compound 2: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-butyloctanoate)

[0038] Compound 3: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-hexyloctanoate)

[0039] Compound 4: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-nonylundecanoate)

[0040] Compound 5: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-butyldodecanoate)

[0041] Compound 6: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonyldodecanoate)

[0042] Compound 7: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate)

[0043] Compound 8: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate)

[0044] Compound 9: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) dioleate

[0045] Compound 10: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(3-nonyldodecanoate)

[0046] Compound 11: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(4-hexyldodecanoate)

[0047] Compound 12: O,O'-((2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)) bis(2-hexyldecyl) disuccinate

[0048] Compound 13: (2-(3-(dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0049] Compound 14: (2-(3-(dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-octyltridecanoate)

[0050] Compound 15: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0051] Compound 16: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0052] Compound 17: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0053] Compound 18: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-hexyldecanoate)

[0054] Compound 19: (2-(2-((dimethylglycyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0055] Compound 20: (2-(2-((methyl-L-prolyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0056] Compound 21: (2-(2-((1-methylpiperidine-4-carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0057] Compound 22: (2-(2-((3-(dimethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0058] Compound 23: (2-(2-((3-(diethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0059] Compound 24: (2-(2-((5-(dimethylamino)pentanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0060] Compound 25: (2-(2-(((3-(dimethylamino)propoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0061] Compound 26: (2-(2-(((3-(dimethylamino)propoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0062] Compound 27: (2-(3-(dimethylamino)propyl)-5-((undecanoyloxy)methyl)-1,3-dioxan-5-yl)methyl 2-nonylundecanoate

[0063] Compound 28: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecyl) bis(carbonate)

[0064] Compound 29: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) di(henicosan-11-yl) bis(carbonate)

[0065] Compound 30: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyloctanoate)

[0066] Compound 31: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyloctanoate)

[0067] Compound 32: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyloctanoate)

[0068] Compound 33: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-hexyloctanoate)

[0069] Compound 34: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0070] Compound 35: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0071] Compound 36: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0072] Compound 37: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-nonylundecanoate)

[0073] Compound 38: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-butyldodecanoate)

[0074] Compound 39: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate)

[0075] Compound 40: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate)

[0076] Compound 41: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate)

[0077] Compound 42: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-octyltridecanoate)

[0078] Compound 43: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate)

[0079] Compound 44: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate)

[0080] Compound 45: (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-dodecyltetradecanoate)

[0081] Compound 46: (2-(3-((3-hydroxypropyl)(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0082] Compound 47: (2-(3-(bis(2-hydroxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0083] Compound 48: (2-(3-(ethyl(2-hydroxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0084] Compound 49: (2-(3-morpholinopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0085] Compound 50: (2-(3-thiomorpholinopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0086] Compound 51: (2-(3-(4-methylpilerazin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0087] Compound 52: (2-(3-(1,4,7,10-tetraoxa-13-azacyclopentadecan-13-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0088] Compound 53: (2-(3-(thiazolidin-3-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0089] Compound 54: (2-(3-(3-oxopiperazin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0090] Compound 55: (2-(3-(ethyl(2-methoxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0091] Compound 56: (2-(3-((2-methoxyethyl)(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0092] Compound 57: (2-(3-(bis(2-methoxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0093] Compound 58: (2-(3-(methyl(oxetan-3-yl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0094] Compound 59: (2-(3-(methyl(1-methylpyrrolidin-3-yl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0095] Compound 60: (2-(3-(azetidin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0096] Compound 61: (2-(3-(pyrrolidine-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0097] Compound 62: (2-(3-(pyrrolidine-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0098] Compound 63: (2-(4-(pyrrolidine-1-yl)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0099] Compound 64: (2-(3-(piperidin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) [[ID=1*]]

[0100] Compound 65: (2-(3-(azepan-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0101] Compound 66: (2-(3-(dipropylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0102] Compound 67: (2-(3-(isobutyl(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0103] Compound 68: (2-(3-(methyl(propyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0104] Compound 69: (2-(3-(isopropyl(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0105] Compound 70: (2-(3-(ethyl(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0106] Compound 71: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0107] Compound 72: (2-(3-(dipropylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0108] Compound 73: (2-(4-(diethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0109] Compound 74: (2-(4-(dipropylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0110] Compound 75: (2-(5-(dimethylamino)pentyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0111] Compound 76: (2-(5-(diethylamino)pentyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0112] Compound 77: (2-(6-(dimethylamino)hexyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0113] Compound 78: (2-(6-(dimethylamino)hexyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)

[0114] Compound 79: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0115] Compound 80: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-heptylnonanoate)

[0116] Compound 81: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyldecanoate)

[0117] Compound 82: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonyldodecanoate)

[0118] Compound 83: (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-undecyltridecanoate)

[0119] Compound 84: (5-(((4-cyclohexylbutanoyl)oxy)methyl)-2-(3-(dimethylamino)propyl)-1,3-dioxan-5-yl)methyl 2-hexyldecanoate

[0120] Compound 85: (2-(3-(dimethylamino)propyl)-5-(((2-hexyldecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl adamantane-1-carboxylate

[0121] Compound 86: (2-(3-(dimethylamino)propyl)-5-(((2-hexyldecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl (1s,4r)-4-pentylcyclohexane-1-carboxylate

[0122] Compound 87: (2-(3-(dimethylamino)propyl)-5-(((2-hexyldecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl (1s,4s)-4-(tert-butyl)cyclohexane-1-carboxylate

[0123] Compound 88: (2-(3-(dimethylamino)propyl)-5-(((2-nonylundecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl (Z)-tetradec-9-enoate

[0124] Compound 89: (2-(3-(dimethylamino)propyl)-5-(((2-nonylundecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl oleate

[0125] Compound 90: (2-(3-(dimethylamino)propyl)-5-(((2-hexyldecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl 2-nonylundecanoate

[0126] Compound 91: 3-(5,5-bis(((2-hexyldecyl)oxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine

[0127] Compound 92: 3-(5,5-bis((nonadecan-10-yloxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine

[0128] Compound 93: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl) bis(2-nonylundecanoate)

[0129] Compound 94: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl) bis(2-hexyldecanoate)

[0130] Compound 95: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyldodecanoate)

[0131] Compound 96: (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate)

[0132] Compound 97: (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyldodecanoate)

[0133] Compound 98: (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyldodecanoate)

[0134] Compound 99: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(4-(heptadecan-9-ylamino)-4-oxobutanoate)

[0135] Compound 100: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(4-(dinonylamino)-4-oxobutanoate)

[0136] Compound 101: di(nonadecan-10-yl) 3,3'-(2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)dipropionate

[0137] Compound 102: bis(2-hexyldecyl) 3,3'-(2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)dipropionate

[0138] Compound 103: (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl) bis(2-hexyloctanoate)

[0139] Compound 104: (((2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene))bis(oxy))bis(propane-3,1-diyl) bis(2-hexyldecanoate)

[0140] Compound 105: (((2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene))bis(oxy))bis(propane-3,1-diyl) bis(2-nonylundecanoate)

[0141] Compound 106: (5-(((3-cyclohexylpropanoyl)oxy)methyl)-2-(3-(dimethylamino)propyl)-1,3-dioxan-5-yl)methyl 2-hexyldecanoate

[0142] Compound 107: (2-(3-(dimethylamino)propyl)-5-((2-(((1R,2S,5R)-2-isopropyl-5-methylcyclohexyl)oxy)acetoxy)methyl)-1,3-dioxan-5-yl)methyl 2-hexyldecanoate

[0143] Compound 108: (2-(3-(dimethylamino)propyl)-5-(((2-hexyldecanoyl)oxy)methyl)-1,3-dioxan-5-yl)methyl 4-((1s,4r)-4-butylcyclohexyl)benzoate

[0144] Compound 109: (5-((((((3R,8R,9R,10S,13S,14R,17S)-10,13-dimethyl-17-((S)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)oxy)methyl)-2-(3-(dimethylamino)propyl)-1,3-dioxan-5-yl)methyl 2-hexyldecanoate

[0145] Compound 110: (2-(4-((3-(dimethylamino)propanoyl)oxy)phenyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0146] Compound 111: (2-(3-((3-(dimethylamino)propanoyl)oxy)phenyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0147] Compound 112: (2-(2-((2-hydroxyethyl)(methyl)amino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0148] Compound 113: (2-(2-(3,4-dihydroxypyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0149] Compound 114: 3-(5,5-bis(((3-((2-hexyldecyl)disulfaneyl)propyl)thio)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine

[0150] Compound 115: 3-(5,5-bis(((2-hexyldecyl)disulfaneyl)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine

[0151] Compound 116: (2-(2-((3-hydroxypropyl)(methyl)amino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0152] Compound 117: (2-(2-(bis(2-hydroxyethyl)amino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0153] Compound 118: (2-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)

[0154] Compound 119: (5-((frac{(2-(adamantan-1-yl)acetoxy)methyl)-2-(3-(dimethylamino)propyl)-1,3-dioxan-5-yl)methyl 2-hexyldecanoate

[0155] In one aspect, the present invention relates to a pH-sensitive cationic lipid.

[0156] The pH-sensitive cationic lipid represented by general formula (I) can be easily produced, for example, by the method specifically shown in the Examples of this specification. By referring to this production method and appropriately selecting raw material compounds, reagents, reaction conditions, etc., a person skilled in the art can easily produce any lipid falling within the scope of general formulas (I) to (III).

[0157] The pH-sensitive cationic lipid of the present invention constituting the lipid nanoparticles of the present invention may be one type only, or may be two or more types. When the pH-sensitive cationic lipid of the present invention constituting the lipid nanoparticles of the present invention is two or more types, the amount of the pH-sensitive cationic lipid of the present invention means the total amount of lipid molecules corresponding to the pH-sensitive cationic lipid of the present invention among the lipid molecules constituting the lipid nanoparticles.

[0158] The higher the ratio of the pH-sensitive cationic lipid of the present invention to the lipid molecules constituting the lipid nanoparticles, the higher the efficiency of uptake of the lipid nanoparticles into target cells. Therefore, in the lipid nanoparticles of the present invention, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles ([amount (mol) of the pH-sensitive cationic lipid of the present invention] / ([total amount (mol) of lipids constituting the lipid nanoparticles]) × 100%) is preferably 20 mol% or more. On the other hand, if the ratio of the pH-sensitive cationic lipid to the lipid molecules constituting the lipid nanoparticles is too high, it may be difficult to sufficiently reduce the particle size. Since lipid nanoparticles have sufficient uptake efficiency into target cells and lipid nanoparticles with a sufficiently small particle size can be obtained, the ratio of the amount of the pH-sensitive cationic lipid of the present invention to the total amount of lipids constituting the lipid nanoparticles in the lipid nanoparticles of the present invention is more preferably 30 mol% or more, even more preferably 30 to 70 mol%, and even more preferably 40 to 60 mol%.

[0159] Among the constituent lipids of the lipid nanoparticles of the present invention, lipids other than the pH-sensitive cationic lipid of the present invention can be lipids generally used in forming liposomes. Examples of such lipids include phospholipids, sterols or sterol derivatives, glycolipids, saturated or unsaturated fatty acids, etc. These can be used alone or in combination of two or more.

[0160] Examples of phospholipids include glycerophospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, phosphalidylcholine, cardiolipin, plasmalogen, ceramide phosphorylglycerol phosphate, and phosphatidic acid; and sphingophospholipids such as sphingomyelin, ceramide phosphorylglycerol, and ceramide phosphorylethanolamine. Phospholipids derived from natural products such as egg yolk lecithin and soybean lecithin can also be used. The fatty acid residues in the glycerophospholipids and sphingophospholipids are not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 24 carbon atoms, with saturated or unsaturated fatty acid residues having 14 to 20 carbon atoms being preferred. Specific examples include acyl groups derived from fatty acids such as lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, arachidonic acid, behenic acid, lignoceric acid, etc. When these glycerolipids or sphingolipids have two or more fatty acid residues, all of the fatty acid residues may be the same group or may be different groups.

[0161] Examples of phospholipids are diphytanoyl phosphatidyl ethanolamine (DPhPE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- These include 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0162] Examples of sterols or sterol derivatives include animal-derived sterols such as cholesterol, cholesterol succinate, lanosterol, dihydrolanosterol, desmosterol, and dihydrocholesterol; plant-derived sterols (phytosterols) such as stigmasterol, sitosterol, β-sitosterol, campesterol, and brassicasterol; and microbial-derived sterols such as zymosterol and ergosterol. Examples of glycolipids include glyceroglycolipids such as sulfoxyribosylglyceride, diglycosyldiglyceride, digalactosyldiglyceride, galactosyldiglyceride, and glycosyldiglyceride; and sphingoglycolipids such as galactosylcerebroside, lactosylcerebroside, and ganglioside. Examples of saturated or unsaturated fatty acids include saturated or unsaturated fatty acids having 12 to 20 carbon atoms such as palmitic acid, oleic acid, stearic acid, arachidonic acid, and myristic acid.

[0163] The constituent lipids of the lipid nanoparticles of the present invention preferably contain a neutral lipid in addition to the pH-sensitive cationic lipid of the present invention, more preferably a phospholipid or a sterol, even more preferably a sterol, and even more preferably cholesterol.

[0164] The lipid nanoparticles according to the present invention preferably contain a polyalkylene glycol-modified lipid as a lipid component. Polyalkylene glycol is a hydrophilic polymer, and by constructing lipid nanoparticles using a polyalkylene glycol-modified lipid as a lipid membrane-constituting lipid, the surface of the lipid nanoparticles can be modified with polyalkylene glycol. Surface modification with polyalkylene glycol may improve the stability of the lipid nanoparticles, such as their blood retention.

[0165] Examples of polyalkylene glycols that can be used include polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and polyhexamethylene glycol. The molecular weight of the polyalkylene glycol is, for example, about 200 to 10,000, for example, about 300 to 10,000, preferably about 500 to 10,000, and more preferably about 1,000 to 5,000. In one embodiment of the present invention, the molecular weight of the polyalkylene glycol is about 200, about 300, about 350, about 400, about 500, about 550, about 750, about 1,000, about 1,500, about 2,000, about 3,000, about 3,500, about 4,000, about 5,000, or about 10,000 Da.

[0166] For example, stearylated polyethylene glycol (e.g., PEG 45 stearate (STR-PEG45)) can be used to modify lipids with polyethylene glycol. Other examples include N-[carbonyl-methoxypolyethylene glycol]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG), N-[carbonyl-methoxypolyethylene glycol]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DSPE-PEG). Polyethylene glycol derivatives such as N-[carbonyl-methoxypolyethylene glycol]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG) can be used.For example, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG2000), N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2000) , N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-750 (DMG-PEG750), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 Polyethylene glycol derivatives such as (DMG-PEG5000), N-[carbonyl-methoxypolyethylene glycol-750]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG750), N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2000), and N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG5000) can also be used, but the polyalkylene glycolated lipid is not limited to these.

[0167] The ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles of the present invention is not particularly limited as long as it is an amount that does not impair the gene expression activity when the lipid nanoparticles of the present invention are used as gene carriers. For example, the ratio of the polyalkylene glycol-modified lipid to the total amount of lipids constituting the lipid nanoparticles is preferably 0.5 to 3 mol%.

[0168] The lipid nanoparticles according to the present invention may contain any one of DSPC, DOPC, and DOPE as a phospholipid, and may contain DMG-PEG as a polyalkylene glycol-modified lipid. For example, lipid nanoparticles of the present invention may comprise 40-60 (e.g., 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60) mol% of pH-sensitive cationic lipid relative to the total lipid content of the lipid nanoparticle; (e.g., 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50) mol% sterol (e.g., cholesterol); 5-15 (e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 50) mol% of the total lipid content of the lipid nanoparticle. 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15) mol% of one of DSPC, DOPC, and DOPE phospholipids; and 1-5 (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 15) mol% of the total lipid content of the lipid nanoparticle.5 or 5) mol % of DMG-PEG (e.g., DMG-PEG2000). In the lipid nanoparticles according to the present invention, the molar ratio of the pH-sensitive cationic lipid, sterol, any one of DSPC, DOPC, and DOPE phospholipids, and DMG-PEG (pH-sensitive cationic lipid / sterol / phospholipid / DMG-PEG) is not particularly limited, and examples thereof include the following: (60 / 31 / 8 / 1), (60 / 31 / 7.5 / 1.5), (60 / 31 / 7 / 2), (60 / 31 / 6.5 / 2.5), (60 / 31 / 6 / 3), (60 / 31 / 5.5 / 3.5), (60 / 31 / 5 / 4), (60 / 31 / 4.5 / 4.5), (60 / 31 / 4 / 5), (60 / 31.5 / 7.5 / 1), (60 / 30.5 / 7.5 / 2), (60 / 30 / 7.5 / 2.5), (60 / 29.5 / 7.5 / 3), (60 / 29 / 7.5 / 3.5), (60 / 28.5 / 7.5 / 4), (60 / 28 / 7.5 / 4.5), (60 / 27.5 / 7.5 / 5), (59 / 32 / 7.5 / 1.5), (58 / 33 / 7.5 / 1.5), (57 / 34 / 7.5 / 1.5), (56 / 35 / 7.5 / 1.5), (55 / 36 / 7.5 / 1.5), (60 / 29 / 10 / 1), (60 / 28.5 / 10 / 1.5), (60 / 28 / 10 / 2), (60 / 27.5 / 10 / 2.5), (60 / 27 / 10 / 3), (60 / 26.5 / 10 / 3.5), (60 / 26 / 10 / 4), (60 / 25.5 / 10 / 4.5), (60 / 25 / 10 / 5), (60 / 27 / 12 / 1), (60 / 27 / 11.5 / 1.5), (60 / 27 / 11 / 2), (60 / 27 / 10.5 / 2.5), (60 / 27 / 9.5 / 3.5), (60 / 27 / 9 / 4), (60 / 27 / 8.5 / 4.5), (60 / 27 / 8 / 5), (59 / 28 / 10 / 3), (58 / 29 / 10 / 3), (57 / 30 / 10 / 3), (56 / 31 / 10 / 3), (55 / 32 / 10 / 3), (50 / 39 / 10 / 1), (50 / 38.5 / 10 / 1.5), (50 / 38 / 10 / 2), (50 / 37.5 / 10 / 2.5), (50 / 37 / 10 / 3), (50 / 36.5 / 10 / 3.5), (50 / 36 / 10 / 4), (50 / 35.5 / 10 / 4).5), (50 / 35 / 10 / 5), (50 / 38.5 / 10.5 / 1), (50 / 38.5 / 9.5 / 2), (50 / 38.5 / 9 / 2.5), (50 / 38.5 / 8.5 / 3), (50 / 38.5 / 8 / 3.5), (50 / 38.5 / 7.5 / 4), (50 / 38.5 / 7 / 4.5), (50 / 38.5 / 6.5 / 5), (51 / 37.5 / 10 / 1.5), (52 / 36.5 / 10 / 1.5), (53 / 35.5 / 10 / 1.5), (54 / 34.5 / 10 / 1.5), (55 / 33.5 / 10 / 1.5), (49 / 39.5 / 10 / 1.5), (48 / 40.5 / 10 / 1.5), (47 / 41.5 / 10 / 1.5), (46 / 42.5 / 10 / 1.5), (45 / 43.5 / 10 / 1.5), (51 / 34 / 10 / 5), (52 / 33 / 10 / 5), (53 / 32 / 10 / 5), (54 / 31 / 10 / 5), (55 / 30 / 10 / 5), (40 / 44 / 15 / 1), (40 / 43.5 / 15 / 1.5), (40 / 43 / 15 / 2), (40 / 42.5 / 15 / 2.5), (40 / 42 / 15 / 3), (40 / 41.5 / 15 / 3.5), (40 / 41 / 15 / 4), (40 / 40.5 / 15 / 4.5), (40 / 40 / 15 / 5), (41 / 39 / 15 / 5), (42 / 38 / 15 / 5), (43 / 37 / 15 / 5), (44 / 36 / 15 / 5), (45 / 35 / 15 / 5), (41 / 41 / 14 / 4), (42 / 42 / 13 / 3), (43 / 43 / 12 / 2), and (44 / 44 / 11 / 1).

[0169] The lipid nanoparticles according to the present invention may contain the following Compound A. Compound A corresponds to Compound T3 in WO2019090359, the entire disclosure of which is incorporated herein by reference.

[0170] The lipid nanoparticles of the present invention contain any one of DOPE, DSPE, and DPPE as a phospholipid, any one of DPPE-PEG, DMPE-PEG, and DSPE-PEG as a polyalkylene glycol-modified lipid, and may also contain compound A. For example, the lipid nanoparticles of the present invention may contain 20-60 (e.g., 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60 mol% of pH-sensitive cationic lipid; 15-50 (e.g., 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5 or 50) mol% sterol (e.g., cholesterol); 5-40 (e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40) mol% of one of DOPE, DSPE, and DPPE phospholipids; 0.5-10 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5) mol% of the total lipid content of the lipid nanoparticle. or 10) mol% of a polyalkylene glycol-modified lipid selected from DPPE-PEG (e.g., DPPE-PEG2000), DMPE-PEG (e.g., DMPE-PEG2000), and DSPE-PEG (e.g., DSPE-PEG2000); and 0.5-10 (e.g., 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5) relative to the total lipid amount of the lipid nanoparticle.May contain 5 or 10 mol% of Compound A. In the lipid nanoparticles according to the present invention, the molar ratio of the pH-sensitive cationic lipid, sterol, any one of the phospholipids DOPE, DSPE, and DPPE, any one of the polyalkylene glycol-modified lipids DPPE-PEG, DMPE-PEG, and DSPE-PEG, and Compound A (pH-sensitive cationic lipid / sterol / phospholipid / polyalkylene glycol-modified lipid / Compound A) is not particularly limited, and examples thereof include the following: (30 / 28 / 30 / 6 / 6), (30 / 29 / 30 / 5.5 / 5.5), (30 / 30 / 30 / 5 / 5), (30 / 31 / 30 / 4.5 / 4.5), (30 / 32 / 30 / 4 / 4), (30 / 33 / 30 / 3.5 / 3.5), (30 / 34 / 30 / 3 / 3), (30 / 35 / 30 / 2.5 / 2.5), (30 / 36 / 30 / 2 / 2), (30 / 37 / 30 / 1.5 / 1.5), (30 / 38 / 30 / 1 / 1), (30 / 30 / 31 / 4.5 / 4.5), (30 / 30 / 32 / 4 / 4), (30 / 30 / 33 / 3.5 / 3.5), (30 / 30 / 34 / 3 / 3), (30 / 30 / 35 / 2.5 / 2.5), (30 / 30 / 36 / 2 / 2), (30 / 30 / 37 / 1.5 / 1.5), (30 / 30 / 38 / 1 / 1), (35 / 25 / 30 / 5 / 5), (34 / 26 / 30 / 5 / 5), (33 / 27 / 30 / 5 / 5), (32 / 28 / 30 / 5 / 5), (31 / 29 / 30 / 5 / 5), (40 / 25 / 25 / 5 / 5), (38 / 26 / 26 / 5 / 5), (36 / 27 / 27 / 5 / 5), (34 / 28 / 28 / 5 / 5), (32 / 29 / 29 / 5 / 5), (50 / 20 / 20 / 5 / 5), (45 / 20 / 25 / 5 / 5), (40 / 25 / 25 / 5 / 5), (35 / 30 / 25 / 5 / 5), (30 / 35 / 25 / 5 / 5), (50 / 21 / 20 / 4.5 / 4.5), (50 / 22 / 20 / 4 / 4), (50 / 23 / 20 / 3.5 / 3.5), (50 / 24 / 20 / 3 / 3), (50 / 25 / 20 / 2.5 / 2.5), (50 / 26 / 20 / 2 / 2), (50 / 27 / 20 / 1.5 / 1.5), (50 / 28 / 20 / 1 / 1), (30 / 50 / 20 / 1 / 1), (30 / 48 / 20 / 2 / 2), (30 / 46 / 20 / 3 / 3), (30 / 44 / 20 / 4 / 4), (30 / 42 / 20 / 5 / 5), (25 / 40 / 25 / 5 / 5), (25 / 40 / 30 / 3 / 2), (25 / 35 / 35 / 3 / 2), (25 / 45 / 25 / 4 / 1), (23 / 35 / 35 / 5 / 2).

[0171] The size of the lipid nanoparticles according to the present invention is preferably 400 nm or less in average particle diameter, more preferably 300 nm or less, even more preferably 200 nm or less, and even more preferably 150 nm or less, since this facilitates high delivery efficiency to cells in vivo. The average particle diameter of the lipid nanoparticles refers to the Z-average particle diameter measured by dynamic light scattering (DLS). Measurement by dynamic light scattering can be performed by a conventional method using a commercially available DLS device or the like.

[0172] The form of the lipid nanoparticles according to the present invention is not particularly limited, and examples thereof include unilamellar liposomes, multilamellar liposomes, spherical micelles, irregular layered structures, etc. The lipid nanoparticles according to the present invention are preferably unilamellar liposomes or multilamellar liposomes when dispersed in an aqueous solvent.

[0173] The lipid nanoparticles according to the present invention preferably contain a target component to be delivered into target cells within the lipid membrane-covered particles. The component contained within the lipid nanoparticles according to the present invention is not particularly limited as long as it has a size that allows it to be contained. The lipid nanoparticles according to the present invention can contain any substance, such as nucleic acids, sugars, peptides, low-molecular-weight compounds, and metal compounds.

[0174] Nucleic acids are preferred as components to be encapsulated in the lipid nanoparticles of the present invention. The nucleic acid may be DNA, RNA, or an analog or derivative thereof (e.g., peptide nucleic acid (PNA), phosphorothioate DNA, etc.). The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention may be a single-stranded nucleic acid, a double-stranded nucleic acid, linear, or cyclic. In one aspect of the present invention, the lipid nanoparticles of the present invention comprise the pH-sensitive cationic lipid of the present invention, a salt or stereoisomer thereof, and a nucleic acid.

[0175] The nucleic acid to be encapsulated in the lipid nanoparticles of the present invention preferably contains a foreign gene for expression in a target cell, and more preferably is a nucleic acid that functions to express the foreign gene in the cell by being incorporated into the cell. The foreign gene may be a gene that is originally contained in the genomic DNA of the target cell, or a gene that is not contained in the genomic DNA. Examples of such nucleic acids include gene expression vectors containing a nucleic acid consisting of a base sequence encoding the gene of interest to be expressed. The gene expression vector may exist as an extrachromosomal gene in the introduced cell, or may be incorporated into the genomic DNA by homologous recombination.

[0176] The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is not particularly limited, and vectors commonly used in gene therapy, etc. can be used. The gene expression vector to be encapsulated in the lipid nanoparticles of the present invention is preferably a nucleic acid vector such as a plasmid vector. The plasmid vector may remain circular, or may be encapsulated in the lipid nanoparticles of the present invention after being pre-cleaved into a linear form. The gene expression vector can be designed by standard methods using commonly used molecular biology tools based on the base sequence information of the gene to be expressed, and can be produced by various known methods.

[0177] The nucleic acid encapsulated in the lipid nanoparticles of the present invention is preferably a functional nucleic acid that controls the expression of a target gene present in a target cell. Examples of such functional nucleic acids include antisense oligonucleotides, antisense DNA, antisense RNA, siRNA, microRNA, and mRNA. Alternatively, the functional nucleic acid may be a plasmid DNA (pDNA) that serves as an siRNA expression vector that expresses siRNA in cells. The siRNA expression vector can be prepared from a commercially available siRNA expression vector, which may also be modified as appropriate. In one aspect of the present invention, the lipid nanoparticles of the present invention comprise the pH-sensitive cationic lipid of the present invention, a salt or stereoisomer thereof, and a nucleic acid, wherein the nucleic acid is siRNA, mRNA, or plasmid DNA.

[0178] The method for producing lipid nanoparticles according to the present invention is not particularly limited, and any method available to those skilled in the art can be employed. For example, lipid nanoparticles according to the present invention can be produced by an alcohol dilution method using a flow channel. This method involves introducing a solution in which a lipid component is dissolved in an alcohol solvent and a solution in which a water-soluble component to be incorporated into the lipid nanoparticles is dissolved in an aqueous solvent through separate flow channels and then merging the resulting solutions to produce lipid nanoparticles. Examples of aqueous solvents used in the alcohol dilution method include buffer solutions such as phosphate buffer, citrate buffer, and phosphate-buffered saline, saline, and cell culture media.

[0179] In one aspect, the present invention relates to a lipid nanoparticle formulation comprising the pH-sensitive cationic lipid of the present invention, its stereoisomer, or a mixture of stereoisomers. In another aspect, the present invention relates to a lipid nanoparticle formulation comprising (i) a sterol or sterol derivative, (ii) a polyalkylene glycol-modified lipid, (iii) a nucleic acid, (iv) a buffer, (v) DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine), and (vi) the pH-sensitive cationic lipid of the present invention, its salt, or a stereoisomer. Examples of sterols or sterol derivatives include cholesterol and sitosterol, preferably cholesterol. Examples of polyalkylene glycol-modified lipids include polyethylene glycol-modified lipids and polypropylene glycol-modified lipids, preferably polyethylene glycol-modified lipids. Examples of nucleic acids include siRNA, pDNA, mRNA, etc., preferably siRNA and mRNA. Examples of buffers include HEPES buffer, phosphate buffer, Tris buffer, etc.

[0180] In the present invention, the lipid nanoparticle formulation may be prepared by suspending lipid nanoparticles in an aqueous solution. The pH of the lipid nanoparticle formulation of the present invention is, for example, 5.5 to 8.5, preferably 6.8 to 8.0, at 25° C. In one aspect, the present invention relates to a resuspended formulation in which the lipid nanoparticle formulation is resuspended by adding water or an aqueous solution.

[0181] The animals to which the lipid nanoparticles of the present invention are administered are not particularly limited and may be humans or non-human animals, including mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quails, and ducks.

[0182] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0183] In the present examples, the following abbreviations were used: THF: tetrahydrofuran (super-dehydrated) (stabilizer-free); LDA: lithium diisopropylamide solution (Sigma-Aldrich, 774766-4X25ML); DMPU: 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone; DMAP: 4-Dimethylaminopyridine; DCM: dichloromethane; EDCI: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (TCI, D1601); TBDPSCl: tert-Butyldiphenylchlorosilane; DMSO: dimethyl sulfoxide; TBAF: tetrabutylammonium fluoride; Pd / C 10%: Palladium 10% on carbon (TCI, P1785); DIPEA: N,N-Diisopropylethylamine; DMF: N,N-dimethylformamide.

[0184] I. Synthesis of Lipids and Raw Materials Raw Material Synthesis Example 1 (Synthesis of α-Alkyl Branched Carboxylic Acid) To THF (50 ml) cooled to -25°C, 23 ml of LDA (1 mol / L) was added and stirred for 60 min. Octanoic acid (1.42 g) dissolved in THF (20 ml) was added, followed by DMPU (1.2 ml). The temperature of the reaction solution was raised to -5°C and stirred for an additional hour. 1-Iodohexane (4.88 g) was added and stirred at room temperature overnight. Saturated aqueous sodium hydrogen sulfate and ethyl acetate were added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, then sodium sulfate was added and dehydrated. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain the desired 2-hexyloctanoic acid (1.75 g). 1H NMR (300 MHz, CDCl) δ 2.41-2.30 (m, 1H), 1.66-1.56 (m, 2H), 1.51-1.42 (m, 2H), 1.28 (brs, 16H), 0.88 (t, J = 6.9 Hz, 6H). 2-Nonylundecanoic acid, 2-butyldodecanoic acid, 2-nonyldodecanoic acid, 2-octyltridecanoic acid, and 2-dodecyltetradecanoic acid were also synthesized in a similar manner.

[0185] Raw Material Synthesis Example 2 (Synthesis of β-Alkyl Branched Carboxylic Acid) (1) NaH (60%, dispersion in paraffin liquid) (216 mg) was dissolved in THF (15 ml) and stirred for 10 minutes under ice-cooling. Benzyl 2-(dimethoxyphosphoryl)acetate (1.13 ml) was added, and the mixture was stirred for an additional 30 minutes under ice-cooling. 10-Nonadecanone (1.27 g) was dissolved in THF (15 ml) and added dropwise to the reaction mixture. The reaction mixture was warmed to room temperature and stirred overnight. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate) to obtain a mixture of the target benzyl 3-nonyldodec-2-enoate and the starting material 10-nonadecanone (928 mg). (2) The mixture obtained in (1) and 10% Pd / C (130 mg) were added to a mixture of THF (4 ml) and MeOH (8 ml). Triethylsilane (796 μl) was then added dropwise and the mixture was stirred for 4 hours. The reaction mixture was filtered, and the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain the desired 3-nonyldodecanoic acid.

[0186] Raw Material Synthesis Example 3 (Synthesis of γ-Alkyl Branched Carboxylic Acid) The target 4-hexyldodecanoic acid was obtained in the same manner as in Raw Material Synthesis Example 1-2, except that 2-hexyldecanal was used instead of 10-nonadecanone in Raw Material Synthesis Example 2(1). 1 H NMR (300 MHz, CDCl3) δ 2.35-2.30 (m, 2H), 1.64-1.56 (m, 2H), 1.33-1.26 (m, 25H), 0.90-0.86 (m, 6H).

[0187] Raw Material Synthesis Example 4 (Synthesis of 2-Hexyldecyl (4-nitrophenyl) carbonate) 2-Hexyldecan-1-ol (2.42 g) and 4-nitrophenyl carbonochloridate (3.02 g) were dissolved in DCM (10 ml). Pyridine (2.42 ml) was added dropwise, and the reaction solution was stirred at room temperature overnight. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain the desired 2-hexyldecyl (4-nitrophenyl) carbonate (3.37 g). 1 H NMR (300 MHz, CDCl3) δ 8.31-8.26 (m, 2H), 7.41-7.36 (m, 2H), 4.19 (d, J = 5.8 Hz 2H), 1.76-1.71 (m, 1H), 1.33-1.29 (m, 24H), 0.91-0.86 (m, 6H).

[0188] Raw Material Synthesis Example 5 (Synthesis of Henicosan-11-yl (4-nitrophenyl) carbonate) The target henicosan-11-yl (4-nitrophenyl) carbonate was obtained in the same manner as in Starting Material Synthesis Example 4, except that henicosan-11-ol was used instead of 2-hexyldecan-1-ol. 1H NMR (300 MHz, CDCl3) δ 8.30-8.25 (m, 2H), 7.41-7.36 (m, 2H), 4.82 (quin, J = 5.6 Hz, 1H), 1.71-1.59 (m, 4H), 1.39-1.26 (m, 32H), 0.88 (t, J = 7.0 Hz, 6H).

[0189] Raw Material Synthesis Example 6 (Synthesis of 4-((2-Hexyldecyl)oxy)-4-oxobutanoic acid) Succinic anhydride (304 mg) and 2-hexyldecan-1-ol (740 mg) were dissolved in toluene (2 ml) and stirred at 100 °C for 6 hours. The solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain the desired 4-((2-hexyldecyl)oxy)-4-oxobutanoic acid (931 mg). 1 H NMR (300 MHz, CDCl3) δ 3.99 (d, J = 5.8 Hz, 2H), 2.72-2.60 (m, 4H), 1.26 (brs, 24H), 0.90-0.86 (m, 6H).

[0190] Lipid Synthesis Example 1 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (1) (2-Phenyl-1,3-dioxane-5,5-diyl)dimethanol (1.12 g), 2-hexyldecanoic acid (2.69 g), and DMAP (61.1 mg) were dissolved in DCM (6.0 ml), EDCI (2.11 g) was added, and the mixture was stirred overnight at room temperature. Saturated aqueous ammonium chloride was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated with sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. (2) Methanol (32 ml), DCM (8.0 ml), and HCl (4 mol / L in 1,4-dioxane, 4.0 ml) were added to the resulting crude product (2.10 g), and the mixture was stirred at room temperature for 4 hours. An excess amount of sodium bicarbonate was added to the reaction solution, and the mixture was concentrated under reduced pressure. Water and ethyl acetate were added to the residue, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated with sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain a crude product, which was then purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain the desired compound, 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-hexyldecanoate) (1.46 g). 1H NMR (300 MHz, CDCl) δ 4.15 (s, 4H), 3.55 (d, J = 6.1 Hz, 4H), 2.87–2.83 (m, 2H), 2.43–2.33 (m, 2H), 1.61–1.43 (m, 8H), 1.26 (brs, 40H), 0.88 (t, J = 7.0 Hz, 12H). (3) 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-hexyldecanoate) (175 mg), 4,4-dimethoxy-N,N-dimethylbutan-1-amine (62.0 mg), p-toluenesulfonic acid monohydrate (65.6 mg), and toluene (1.0 ml) were placed in a vessel equipped with a calcium chloride tube and stirred at 85 °C overnight. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to obtain the desired (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (131 mg). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.6 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.9 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.9 Hz, 2H), 2.37-2.25 (m, 4H), 2.22 (s, 6H), 1.72-1.40 (m, 12H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 710.6, Measured m / z = 710.6.

[0191] Lipid Synthesis Example 2 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyloctanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyloctanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-butyloctanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.49 (t, J = 4.5 Hz, 1H), 4.36 (s 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.84 (s, 2H), 3.60 (d, J = 11.7 Hz, 2H), 2.36-2.25 (m, 4H), 2.22 (s, 6H), 1.67-1.42 (m, 12H), 1.25 (brs, 24H), 0.85-0.90 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 598.5, Measured m / z = 598.5.

[0192] Lipid Synthesis Example 3 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyloctanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyloctanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-hexyloctanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.5 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.84 (s, 2H), 3.60 (d, J = 11.7 Hz, 2H), 2.38-2.24 (m, 4H), 2.21 (s, 6H), 1.62-1.45 (m, 12H), 1.25 (brs, 32H) 0.90-0.87 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 654.6, Measured m / z = 654.6.

[0193] Lipid Synthesis Example 4 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-nonylundecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.4 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.8 Hz, 2H), 2.37-2.24 (m, 4H), 2.21 (s, 6H), 1.65-1.40 (m, 12H), 1.25 (brs, 56H), 0.88 (t, J = 6.45 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 822.8, Measured m / z = 822.8.

[0194] Lipid Synthesis Example 5 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyldodecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-butyldodecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-butyldodecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.7 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.7 Hz, 2H), 2.37-2.24 (m, 4H), 2.21 (s, 6H), 1.66-1.40 (m, 12H), 1.30-1.25 (m, 40H), 0.90-0.86 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 710.6, Measured m / z = 710.6.

[0195] Lipid Synthesis Example 6 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonyldodecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonyldodecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-nonyldodecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 2.35-2.24 (m, 4H), 2.22 (s, 6H), 1.57-1.45 (m, 12H), 1.25 (brs, 60H), 0.90-0.87 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 850.8, Measured m / z = 850.8.

[0196] Lipid Synthesis Example 7 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyltridecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-octyltridecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.5Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.8 Hz, 2H), 2.37-2.25 (m, 4H), 2.22 (s, 6H), 1.67-1.43 (m, 12H), 1.25 (brs, 60H), 0.90-0.87 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 850.8, Measured m / z = 850.8.

[0197] Lipid Synthesis Example 8 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-dodecyltetradecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 2-dodecyltetradecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.5Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 2.39-2.24 (m, 4H), 2.22 (s, 6H), 1.62-1.43 (m, 12H), 1.25 (brs, 80H), 0.90-0.86 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 990.9, Measured m / z = 990.9.

[0198] Lipid Synthesis Example 9 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) dioleate) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) dioleate was obtained in the same manner as in Lipid Synthesis Example 1, except that oleic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 5.36-5.32 (m, 4H), 4.56 (t, J = 4.0 Hz, 1H), 4.34 (s, 2H), 3.88 (d, J = 12.0 Hz, 2H), 3.85 (s, 2H), 3.60 (d, J = 12.0 Hz, 2H), 3.10-3.05 (m, 24H), 2.82 (s, 6H), 2.31 (td, J = 7.3 Hz, 5.9 Hz, 4H), 2.02-1.98 (m, 8H), 1.94-1.86 (m, 2H), 1.72-1.62 (m, 4H), 1.30-1.27 (m, 42H), 0.88 (t, J = 6.5 Hz, 6H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 762.7, Measured m / z = 762.7.

[0199] Lipid Synthesis Example 10 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(3-nonyldodecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(3-nonyldodecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 3-nonyldodecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.49 (t, J = 4.2 Hz, 1H), 4.35 (s, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.82 (s, 2H), 3.59 (d, J = 11.8 Hz, 2H), 2.31-2.22 (m, 12H), 1.81-1.63 (m, 6H), 1.25 (brs, 64H), 0.88 (t, J = 7.0 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 850.8, Measured m / z = 850.8.

[0200] Lipid Synthesis Example 11 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(4-hexyldodecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(4-hexyldodecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 4-hexyldodecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1 H NMR (300 MHz, CDCl3) δ 4.50 (t, J = 4.3 Hz, 1H), 4.35 (s, 2H), 3.93 (d, J = 12.0 Hz, 2H), 3.84 (s, 2H), 3.60 (d, J = 12.0 Hz, 2H), 2.36-2.25 (m, 12H), 1.65-1.53 ​​(m, 8H), 1.33-1.24 (m, 50H), 0.88 (t, J = 6.9 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 766.7, Measured m / z = 766.7.

[0201] Lipid Synthesis Example 12 (Synthesis of O,O'-((2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)) bis(2-hexyldecyl) disuccinate) The target O,O'-((2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene)) bis(2-hexyldecyl) disuccinate was obtained in the same manner as in Lipid Synthesis Example 1, except that 4-((2-hexyldecyl)oxy)-4-oxobutanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 4.51 (t, J = 4.2 Hz, 1H), 4.39 (s, 2H), 3.98 (dd, J = 5.9 Hz, 2.0 Hz, 4H), 3.91 (d, J = 11.8 Hz, 2H), 3.87 (s, 2H), 3.59 (d, J = 11.8 Hz, 2H), 2.65-2.63 (m, 8H),2.55-2.50 (m, 2H), 2.41 (s, 6H), 1.67-1.62 (m, 6H), 1.27 (brs, 48H), 0.88 (t, J = 6.7 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 882.7, Measured m / z = 882.7.

[0202] Lipid Synthesis Example 13 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that ((4R,5R)-2-phenyl-1,3-dioxolane-4,5-diyl)dimethanol was used instead of (2-phenyl-1,3-dioxane-5,5-diyl)dimethanol in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 5.19-5.14 (m, 0.3H), 5.10 (t, J = 4.7 Hz, 0.4H), 5.00 (t, J = 4.7 Hz, 0.15H), 4.90 (t, J = 4.7 Hz, 0.15H), 4.67-4.61 (m, 0.65H), 4.41-4.34 (m, 0.35H), 4.26-4.03 (m, 4H), 3.86-3.61 (m, 1H), 2.49-2.27 (m, 4H), 2.23-2.22 (m, 6H), 1.66-1.42 (m, 12H), 1.25 (brs, 40H), 0.88 (t, J = 7.0 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 696.6, Measured m / z = 696.6.

[0203] Lipid Synthesis Example 14 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-octyltridecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxolane-4,5-diyl)bis(methylene) bis(2-octyltridecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that ((4R,5R)-2-phenyl-1,3-dioxolane-4,5-diyl)dimethanol was used instead of (2-phenyl-1,3-dioxane-5,5-diyl)dimethanol and 2-octyltridecanoic acid was used instead of 2-hexyldecanoic acid in Lipid Synthesis Example 1(1). 1H NMR (300 MHz, CDCl3) δ 5.18-5.14 (m, 0.3H), 5.10 (t, J = 4.6 Hz, 0.4H), 5.02-4.88 (m, 0.3H), 4.67-4.61 (m, 0.65H), 4.41-4.34 (m, 0.35H), 4.26-4.03 (m, 4H), 3.86-3.61 (m, 1H), 2.49-2.27 (m, 4H), 2.23-2.22 (m, 6H), 1.66-1.42 (m, 12H), 1.25 (brs, 60H), 0.88 (t, J = 7.2 Hz, 12H). Mass spectrometry (ESI) Calculated value [M+H] + m / z = 836.8, Measured m / z = 836.8.

[0204] Lipid Synthesis Example 15 (Synthesis of (2-(3-(Diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(diethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 4,4-diethoxy-N,N-diethylbutan-1-amine was used instead of 4,4-dimethoxy-N,N-dimethylbutan-1-amine in Lipid Synthesis Example 1(3). 1H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 5.1 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.8 Hz, 2H), 2.51 (q, J = 7.0 Hz, 4H), 2.44 (t, J = 6.9 Hz, 2H), 2.37-2.28 (m, 2H), 1.61-1.43 (m, 12H), 1.25 (brs, 40H), 1.04 (t, J = 7.1 Hz, 6H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 738.7, Measured m / z = 738.7.

[0205] Lipid Synthesis Example 16 (Synthesis of (2-(2-(Dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-(dimethylamino)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 3,3-diethoxy-N,N-dimethylpropan-1-amine was used instead of 4,4-dimethoxy-N,N-dimethylbutan-1-amine in Lipid Synthesis Example 1(3). 1 H NMR (300 MHz, CDCl3) δ 4.56 (t, J = 4.7 Hz, 1H), 4.35 (s, 2H), 3.91 (d, J = 11.5 Hz, 2H), 3.83 (s, 2H), 3.62 (d, J = 11.5 Hz, 2H), 2.43-2.30 (m, 4H), 2.25 (s, 6H), 1.85-1.79 (m, 2H), 1.63-1.53 ​​(m, 4H), 1.50-1.40 (m, 4H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] +m / z = 696.6, Measured m / z = 696.6.

[0206] Lipid Synthesis Example 17 (Synthesis of (2-(2-(Pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-(pyrrolidin-1-yl)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 1-(3,3-diethoxypropyl)pyrrolidine was used instead of 4,4-dimethoxy-N,N-dimethylbutan-1-amine in Lipid Synthesis Example 1(3). 1 H NMR (300 MHz, CDCl3) δ 4.57 (t, J = 4.9 Hz, 1H), 4.35 (s, 2H), 3.91 (d, J = 11.6 Hz, 2H), 3.83 (s, 2H), 3.61 (d, J = 11.6 Hz, 2H), 2.61-2.54 (m, 6H), 2.39-2.28 (m, 2H), 1.92-1.85 (m, 2H), 1.82-1.77 (m, 4H), 1.63-1.53 ​​(m, 4H), 1.50-1.40 (m, 4H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) Calculated value [M+H] + m / z = 722.6, Measured m / z = 722.6.

[0207] Lipid Synthesis Example 18 (Synthesis of (9-Methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (9-methyl-1,5-dioxa-9-azaspiro[5.5]undecane-3,3-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 1, except that 1-methylpiperidin-4-one was used instead of 4,4-dimethoxy-N,N-dimethylbutan-1-amine in Lipid Synthesis Example 1(3). 1 H NMR (300 MHz, CDCl3) δ4.10 (s, 4H), 3.74 (s, 4H), 2.44-2.41 (m, 4H), 2.37-2.31 (m, 2H), 2.29 (s, 3H), 1.93-1.89 (m, 4H), 1.59-1.41 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 708.6, Measured m / z = 708.6.

[0208] Raw Material Synthesis Example 7 (Synthesis of 3-((tert-Butyldiphenylsilyl)oxy)propanal) (1) 1,3-Propanediol (2.28 g) and pyridine (3.22 ml) were dissolved in DCM (20 ml), TBDPSCl (5.50 g) was added, and the mixture was stirred overnight at room temperature. Saturated aqueous ammonium chloride was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain 3-((tert-butyldiphenylsilyl)oxy)propan-1-ol (6.65 g). (2) 3-((tert-Butyldiphenylsilyl)oxy)propan-1-ol (3.15 g), DMSO (7.10 ml), and triethylamine (6.93 ml) were dissolved in DCM (30 ml) and stirred under ice cooling. After 10 minutes, sulfur trioxide pyridine complex (4.77 g) was added, and the reaction solution was warmed to room temperature and stirred overnight. Saturated aqueous ammonium chloride was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 3-((tert-butyldiphenylsilyl)oxy)propanal (2.37 g). 1 H NMR (300 MHz, CDCl3) δ 9.82 (t, J = 2.2 Hz, 1H), 7.77-7.64 (m, 4H), 7.44-7.36 (m, 6H), 4.02 (t, J = 6.0 Hz, 2H), 2.60 (td, J = 6.0 Hz, 2.2 Hz, 2H), 1.04 (s, 9H).

[0209] Raw Material Synthesis Example 8 (Synthesis of (2-(2-Hydroxyethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (1) 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-hexyldecanoate) (2.76 g), 3-((tert-butyldiphenylsilyl)oxy)propanal (1.69 g), p-toluenesulfonic acid monohydrate (85.6 mg), and toluene (30 ml) were added to a vessel equipped with a calcium chloride tube and stirred overnight at 80°C. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (3.02 g). (2) (2-(2-((tert-Butyldiphenylsilyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (1.36 g) and acetic acid (2.57 ml) were mixed with TBAF (1.0 mol / L in THF, 30 ml) and stirred at room temperature for 3 hours. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(2-hydroxyethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (692 mg). 1H NMR (300 MHz, CDCl3) δ 4.70 (t, J = 4.7 Hz, 1H), 4.38 (s, 2H), 3.94 (d, J = 11.8 Hz, 2H), 3.84 (s, 2H), 3.78 (q, J = 5.6 Hz, 2H), 3.64 (d, J = 11.8 Hz, 2H), 2.40-2.29 (m, 2H), 2.19 (t, J = 5.6 Hz, 1H), 1.91 (q, J = 4.9 Hz, 2H), 1.65-1.41 (m, 8H), 1.25 (brs, 40H), 0.89-0.86 (m, 12H).

[0210] Lipid Synthesis Example 19 (Synthesis of (2-(2-((Dimethylglycyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (2-(2-Hydroxyethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (134 mg), N,N-dimethylglycine (41.3 mg), and DMAP (2.44 mg) were dissolved in DCM (2 ml), and EDCI (76.7 mg) was added and stirred overnight at room temperature. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to obtain (2-(2-((dimethylglycyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (92.0 mg). 1H NMR (300 MHz, CDCl3) δ4.58 (t, J = 5.2 Hz, 1H), 4.35 (s, 2H), 4.24 (t, J = 6.5 Hz, 2H), 3.92 (d, J = 11.9 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.9 Hz, 2H), 3.16 (s, 2H), 2.42-2.28 (m, 8H), 1.98 (q, J = 6.5 Hz, 2H), 1.63-1.40 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 754.6, Measured m / z = 754.6.

[0211] Lipid synthesis example 20 (synthesis of (2-(2-((Methyl-L-prolyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-((methyl-L-prolyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 19, except that N-methyl-L-proline monohydrate was used instead of N,N-dimethylglycine in Lipid Synthesis Example 19. 1H NMR (300 MHz, CDCl3) δ4.58 (t, J = 5.0 Hz, 1H), 4.35 (s, 2H), 4.24 (td, J = 6.5 Hz, 1.8 Hz, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 3.16-3.10 (m, 1H),2.97-2.92 (m, 1H), 2.40 (s, 3H), 2.37-2.27 (m, 3H), 2.20-2.10 (m, 1H), 2.02-1.86 (m, 4H), 1.84-1.72 (m, 1H), 1.63-1.40 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 780.6, Measured m / z = 780.6.

[0212] Lipid Synthesis Example 21 (Synthesis of (2-(2-((1-Methylpiperidine-4-carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-((1-methylpiperidine-4-carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 19, except that 1-methylpiperidine-4-carboxylic acid hydrochloride was used instead of N,N-dimethylglycine in Lipid Synthesis Example 19. 1H NMR (300 MHz, CDCl3) δ4.57 (t, J = 5.3 Hz, 1H), 4.35 (s, 2H), 4.18 (t, J = 6.5 Hz, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 2.85-2.81 (m, 2H), 2.39-2.24 (m, 6H), 2.06-1.93 (m, 6H), 1.85-1.41 (m, 10H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 794.7, Measured m / z = 794.7.

[0213] Lipid Synthesis Example 22 (Synthesis of (2-(2-((3-(Dimethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-((3-(dimethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 19, except that 3-(dimethylamino)propanoic acid hydrochloride was used instead of N,N-dimethylglycine in Lipid Synthesis Example 19. 1H NMR (300 MHz, CDCl3) δ4.58 (t, J = 5.1 Hz, 1H), 4.35 (s, 2H), 4.19 (t, J = 6.4 Hz, 2H), 3.92 (d, J = 11.8 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.8 Hz, 2H), 2.64-2.59 (m, 2H), 2.50-2.45 (m, 2H), 2.39-2.28 (m, 2H), 2.24 (s, 6H), 1.95 (q, J = 5.4 Hz, 2H), 1.66-1.40 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 768.6, Measured m / z = 768.6.

[0214] Lipid Synthesis Example 23 (Synthesis of (2-(2-((3-(Diethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-((3-(diethylamino)propanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 19, except that 3-(diethylamino)propionic acid hydrochloride was used instead of N,N-dimethylglycine in Lipid Synthesis Example 19. 1H NMR (300 MHz, CDCl3) δ4.58 (t, J = 5.2 Hz, 1H), 4.35 (s, 2H), 4.18 (t, J = 6.5 Hz, 2H), 3.92 (d, J = 12.0 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 12.0 Hz, 2H), 2.80 (t, J = 7.7 Hz, 2H), 2.52 (q, J = 7.2 Hz, 4H), 2.45 (t, J = 7.7 Hz, 2H), 2.36-2.30 (m, 2H), 1.95 (q, J = 6.6 Hz, 2H), 1.66-1.40 (m, 8H), 1.25 (brs, 40H), 1.03 (t, J = 7.2 Hz, 6H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 796.7, Measured m / z = 796.7.

[0215] Lipid Synthesis Example 24 (Synthesis of (2-(2-((5-(Dimethylamino)pentanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(2-((5-(dimethylamino)pentanoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 19, except that 5-(dimethylamino)pentanoic acid hydrochloride was used instead of N,N-dimethylglycine in Lipid Synthesis Example 19. 1H NMR (300 MHz, CDCl3) δ4.58 (t, J = 5.3 Hz, 1H), 4.35 (s, 2H), 4.17 (t, J = 6.4 Hz, 2H), 3.92 (d, J = 12.0 Hz, 2H), 3.83 (s, 2H), 3.61 (d, J = 12.0 Hz, 2H), 2.51-2.46 (m, 2H), 2.39 (s, 6H), 2.37-2.32 (m, 4H), 1.95 (q, J = 6.5 Hz, 2H), 1.66-1.40 (m, 12H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 796.7, Measured value m / z = 796.7.

[0216] Lipid Synthesis Example 25 (Synthesis of (2-(2-(((3-(Dimethylamino)propoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (1) 4-Nitrophenyl carbonochloridate (55.6 mg) and triethylamine (41.5 μL) were dissolved in DCM (2 mL). (2-(2-Hydroxyethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (154 mg) was dissolved in DCM (1 mL) and added dropwise to the reaction solution. After stirring at room temperature for 4 hours, saturated aqueous ammonium chloride was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(2-(((4-nitrophenoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (87.1 mg). (2) (2-(2-(((4-Nitrophenoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (41.7 mg) and triethylamine (15.3 μl) were dissolved in DCM (1 ml), and 3-(dimethylamino)propan-1-ol (18.8 μl) was added. After stirring overnight at room temperature, saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to give (2-(2-(((3-(dimethylamino)propoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (16.4 mg). 1H NMR (300 MHz, CDCl3) δ4.63 (t, J = 5.2 Hz, 1H), 4.35 (s, 2H), 4.26-4.17 (m, 4H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.61 (d, J = 11.7 Hz, 2H), 2.43-2.29 (m, 4H), 2.39 (s, 6H), 1.99 (q, J = 6.5 Hz, 2H), 1.87 (quin, J = 7.2 Hz, 2H), 1.65-1.40 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) Calculated [M+H] + m / z = 798.6, Measured m / z = 798.6.

[0217] Lipid Synthesis Example 26 (Synthesis of (2-(2-(((2-(Dimethylamino)ethyl)carbamoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (2-(2-(((4-Nitrophenoxy)carbonyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (41.7 mg) and triethylamine (15.3 μl) were dissolved in DCM (1 ml), and N1,N1-dimethylethane-1,2-diamine (6.50 μl) was added. After stirring overnight at room temperature, saturated aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine, and then sodium sulfate was added for dehydration. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to give (2-(2-(((2-(dimethylamino)ethyl)carbamoyl)oxy)ethyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (22.6 mg). 1H NMR (300 MHz, CDCl3) δ5.21 (brs, 1H), 4.59 (t, J = 5.2 Hz, 1H), 4.36 (s, 2H), 4.15 (t, J = 6.2 Hz, 2H), 3.92 (d, J = 11.9 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.9 Hz, 2H), 3.26 (q, J = 5.6 Hz, 2H), 2.47-2.29 (m, 4H), 2.25 (s, 6H), 1.99 (q, J = 5.8 Hz, 2H), 1.63-1.40 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Calculated by mass spectrometry (ESI) [M+H] + m / z = 783.6, Measured value m / z = 783.6.

[0218] Lipid Synthesis Example 27 (Synthesis of (2-(3-(Dimethylamino)propyl)-5-((undecanoyloxy)methyl)-1,3-dioxan-5-yl)methyl 2-nonylundecanoate) (1) Pentaerythritol (1.36 g), undecanoic acid (931 mg), and DMAP (30.5 mg) were added to DMF (20 ml), and the reaction mixture was heated to 80°C until the pentaerythritol was completely dissolved. After cooling to room temperature, EDCI (1.15 g) was added and the mixture was stirred overnight. A saturated aqueous solution of ammonium chloride was added to the reaction mixture, and the mixture was extracted three times with a hexane:ethyl acetate (50%, 50%) mixed solvent. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (elution solvent: hexane / ethyl acetate) to obtain 3-hydroxy-2,2-bis(hydroxymethyl)propyl undecanoate. (2) 3-hydroxy-2,2-bis(hydroxymethyl)propyl undecanoate (183 mg), 4,4-dimethoxy-N,N-dimethylbutan-1-amine (96.8 mg), p-toluenesulfonic acid monohydrate (1.14 mg), HCl (4.0 mol / L in dioxan, 150 μL), and DMF (2 mL) were added to a vessel equipped with a calcium chloride tube and stirred overnight at 80°C. The reaction mixture was cooled to room temperature, and then 2-nonylundecanoic acid (188 mg), DMAP (7.33 mg), and EDCI (138 mg) were added and stirred overnight. Aqueous sodium hydroxide (5.0 mol / L) was added to the reaction mixture, which was then extracted three times with a hexane:ethyl acetate (50%, 50%) mixed solvent. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to give (2-(3-(dimethylamino)propyl)-5-((undecanoyloxy)methyl)-1,3-dioxan-5-yl)methyl 2-nonylundecanoate (74.4 mg). 1H NMR (300 MHz, CDCl3) δ 4.56 (t, J = 3.8 Hz, 1H), 4.34 (s, 2H), 3.93-3.85 (m, 4H), 3.60 (d, J = 11.8 Hz, 2H), 3.12-3.06 (m, 2H), 2.82 (s, 6H), 2.83-2.28 (m, 3H), 1.95-1.87 (m, 2H), 1.75-1.41 (m, 8H), 1.25 (brs, 42H), 0.88 (t, J = 6.9 Hz, 9H). Mass spectrometry (ESI) calcd. [M+H] + m / z = 696.6, Measured value m / z = 696.6.

[0219] Lipid Synthesis Example 28 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecyl) bis(carbonate)) Pentaerythritol (136 mg) was added to DMF (5 ml) and heated to 80°C until the pentaerythritol was completely dissolved. To the reaction solution, HCl (4.0 mol / L in dioxan, 100 μl), p-toluenesulfonic acid monohydrate (1.14 mg), and 4,4-dimethoxy-N,N-dimethylbutan-1-amine (64.5 mg) were added and stirred overnight at 80°C. After the reaction solution was cooled to room temperature, pyridine (484 μl), DMAP (48.9 mg), and 2-hexyldecyl (4-nitrophenyl) carbonate (1.63 g) were added and stirred overnight at room temperature. Aqueous sodium hydroxide (5.0 mol / L) was added to the reaction solution, which was then extracted three times with a hexane:ethyl acetate (50%, 50%) mixed solvent. The organic layer was washed with saturated brine and dehydrated by adding sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to give (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecyl) bis(carbonate) (116 mg). 1 H NMR (300 MHz, CDCl3) δ 4.55 (t, J = 4.3 Hz, 1H), 4.42 (s, 2H), 4.02 (d, J = 5.7 Hz, 4H), 3.98-3.95 (m, 4H), 3.64 (d, J = 11.9 Hz, Mass spectrometry (ESI) Calculated value [M+H] + m / z = 770.7, Measured m / z = 770.7.

[0220] Lipid Synthesis Example 29 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) di(henicosan-11-yl) bis(carbonate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) di(henicosan-11-yl) bis(carbonate) was obtained in the same manner as in Lipid Synthesis Example 28, except that henicosan-11-yl (4-nitrophenyl) carbonate was used instead of 2-hexyldecyl (4-nitrophenyl) carbonate in Lipid Synthesis Example 28. 1 H NMR (300 MHz, CDCl3) δ 4.68-4.64 (m, 2H), 4.51 (brs, 1H), 4.42 (s, 2H), 3.99-3.95 (m, 4H), 3.65 (d, J = 11.8 Hz, 2H), 2.43 (brs, 2H), 2.34 (s, 6H), 1.66-1.55 (m, 12H), 1.25 (brs, 64H), 0.88 (t, J = 6.9 Hz, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 910.8, Measured m / z = 910.8.

[0221] 2-heptylnonanoic acid, 2-octyldecanoic acid, and 2-undecyltridecanoic acid were also synthesized in the same manner as in Raw Material Synthesis Example 1. Furthermore, 4-((tert-butyldiphenylsilyl)oxy)butanal, 5-((tert-butyldiphenylsilyl)oxy)pentanal, 6-((tert-butyldiphenylsilyl)oxy)hexanal, and 7-((tert-butyldiphenylsilyl)oxy)heptanal were also synthesized in the same manner as in Raw Material Synthesis Example 7. In addition, (2-(3-hydroxypropyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-hexyldecanoate), (2-(3-hydroxypropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(4-hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(5-hydroxypentyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate) and (2-(6-hydroxyhexyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate) were also synthesized.

[0222] Raw Material Synthesis Example 9 (Synthesis of (2-(4-Hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (1) Pentaerythritol (1.04 g), pyridine (16 ml), and DMAP (910 mg) were added to DMF (600 ml) and heated in a 70°C water bath until the pentaerythritol was completely dissolved. Acetic anhydride (14 ml) was added dropwise to the reaction solution, and the mixture was stirred overnight at room temperature. The solvent was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: DCM / methanol) to obtain a 1:1 mixture of 2,2-bis(hydroxymethyl)propane-1,3-diyl diacetate and DMF. (2) A mixture of 2,2-bis(hydroxymethyl)propane-1,3-diyl diacetate (1.17 g), 5-((tert-butyldiphenylsilyl)oxy)pentanal (1.36 g), and p-toluenesulfonic acid monohydrate (38.0 mg) was added to toluene (20 ml) and stirred overnight at 85°C. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then sodium sulfate was added for dehydration. After filtration, the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(4-((tert-butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) diacetate (1.32 g). 1H NMR (300 MHz, CDCl3) δ7.67-7.64 (m, 4H), 7.45-7.33 (m, 6H), 4.43 (t, J = 5.1 Hz, 1H), 4.36 (s, 2H), 3.93 (d, J = 11.7 Hz, 2H), 3.83 (s, (3) (2-(4-((tert-Butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) Diacetate (1.32 g) and potassium carbonate (553 mg) were added to methanol (10 ml) and stirred at room temperature for 5 hours. An excess amount of saturated aqueous ammonium chloride was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, then sodium sulfate was added and the mixture was dehydrated. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(4-((tert-butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)dimethanol (1.17 g). 1H NMR (300 MHz, CDCl3) δ7.67-7.64 (m, 4H), 7.45-7.33 (m, 6H), 4.43 (t, J = 5.1 Hz, 1H), 4.03-3.98 (m, 4H), 3.65 (t, J = 6.6 Hz, 2H), 3.54-3.48 (m, 4H), 2.12 (brs, 1H), 2.02 (brs, 1H), 1.63-1.53 ​​(m, 4H), 1.50-1.41 (m, 2H), 1.04 (s, 9H). (4) (2-(4-((tert-Butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)dimethanol(233 mg), 2-hexyldecanoic acid (356 mg), triethylamine (169 μL), and DMAP (6.22 mg) were dissolved in DCM (2 ml), EDCI (293 mg) was added, and the mixture was stirred overnight at room temperature. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain (2-(4-((tert-butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (337 mg). 1H NMR (300 MHz, CDCl3) δ7.67-7.64 (m, 4H), 7.42-7.35 (m, 6H), 4.42 (t, J = 4.8 Hz, 1H), 4.35 (s, 2H), 3.91 (d, J = 11.7 Hz, 2H), 3.83 (s, (5) (2-(4-((tert-Butyldiphenylsilyl)oxy)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (337 mg) was dissolved in THF (1 ml), and TBAF (1.0 mol / L in THF, 1.5 ml) was added and stirred at room temperature for 4 hours. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain the desired (2-(4-hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (226 mg).

[0223] (2-(4-Hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-heptylnonanoate), (2-(4-hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyldecanoate), (2-(4-hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-nonyldodecanoate) and (2-(4-hydroxybutyl)-1,3-dioxane-5,5-diyl)bis(methylene)bis(2-undecyltridecanoate) were synthesized in a similar manner.

[0224] Raw Material Synthesis Example 10 (Synthesis of (2-(3-Oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (2-(3-Hydroxypropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (3.48 g), DMSO (3.62 ml), and triethylamine (3.53 ml) were dissolved in DCM (25 ml) and stirred under ice cooling. After 10 minutes, sulfur trioxide pyridine complex (2.44 g) was added, and the reaction solution was warmed to room temperature and stirred for 4 hours. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain the desired (2-(3-oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (2.83 g). 1H NMR (300 MHz, CDCl3) δ 9.76 (t, J = 1.2 Hz, 1H), 4.56 (t, J = 4.5 Hz, 1H), 4.34 (s, 2H), 3.91 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.7 Hz, 2H), 2.58 (dt, J = 9.0 Hz, 1.2 Hz, 2H), 2.37-2.30 (m, 2H), 1.97 (dt, J = 7.2 Hz, 4.5 Hz, 2H), 1.74-1.39 (m, 8H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H).

[0225] (2-(3-Oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(5-oxopentyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(6-oxohexyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate), (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate), (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-heptylnonanoate), (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-octyldecanoate), (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonyldodecanoate), and (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-undecyltridecanoate) were synthesized in a similar manner.

[0226] Lipid Synthesis Examples 30 to 45 The lipids in Table 1 below were synthesized by appropriately replacing 2-hexyldecanoic acid in Lipid Synthesis Example 1(1) and 4,4-dimethoxy-N,N-dimethylbutan-1-amine in Lipid Synthesis Example 1(3) with other raw materials.

[0227] Lipid Synthesis Example 46 (Synthesis of (2-(3-((3-Hydroxypropyl)(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) (2-(3-Oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (102 mg) and 3-(methylamino)propan-1-ol (14.7 mg) were dissolved in THF (1 ml), and sodium triacetoxyborohydride (44.5 mg) was added. The mixture was stirred at room temperature for 4 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to obtain the desired (2-(3-((3-hydroxypropyl)(methyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) (68.7 mg). 1 H NMR (300 MHz, CDCl3) δ 4.48 (t, J = 4.2Hz, 1H), 4.35 (s, 2H), 3.91 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.80 (t, J = 5.1 Hz, 2H), 3.60 (d, J = 11.7 Hz, 2H), 2.61 (t, J = 5.1 Hz, 2H), 2,42-2.30 (m, 4H), 2.26 (s, 3H), 1.72-1.43 (m, 14H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 754.7, Measured m / z = 754.7.

[0228] Lipid Synthesis Examples 47 and 48 The lipids shown in Table 2 below were synthesized by replacing 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 with an amino alcohol shown in Table 2 below.

[0229] Lipid Synthesis Example 49 (Synthesis of (2-(3-morpholinopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-morpholinopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that morpholine was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46. 1 H NMR (300 MHz, CDCl3) δ4.48 (t, J = 4.5 Hz, 1H), 4.35 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.84 (s, 2H), 3.70 (t, J = 4.5 Hz, 4H), 3.61 (d, J Mass spectrometry (ESI) calculated values [M+H] + m / z = 752.6, Measured m / z = 752.6.

[0230] Lipid Synthesis Examples 50 to 52 The lipids in Table 3 below were synthesized by replacing 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 with a cyclic amine in Table 3 below.

[0231] Lipid Synthesis Example 55 (Synthesis of (2-(3-(ethyl(2-methoxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(ethyl(2-methoxyethyl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that N-ethyl-2-methoxyethan-1-amine was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46. 1 H NMR (300 MHz, CDCl3) δ4.47 (t, J = 4.5 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 3.44 (t, J = 6.0 Hz, 2H), 3.43 (s, 3H), 2.63 (t, J = 6.0 Hz, 2H), 2.55 (q, J = 6.9 Hz, 2H), 2.47 (t, J = 7.2 Hz, 2H), 2.36-2.30 (m, 2H), 1.62-1.38 (m. 12H), 1.25 (brs, 40H), 1.00 (t, J = 6.9 Hz, 3H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 768.7, Measured m / z = 768.7.

[0232] Lipid Synthesis Examples 56 to 58 The lipids in Table 4 below were synthesized by replacing 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 with an ether amine in Table 4 below.

[0233] Lipid Synthesis Example 59 (Synthesis of (2-(3-(methyl(1-methylpyrrolidin-3-yl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(methyl(1-methylpyrrolidin-3-yl)amino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that N,1-dimethylpyrrolidin-3-amine was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46. 1 H NMR (300 MHz, CDCl3) δ4.47 (t, J = 4.2 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz, 2H), 3.08-2.98 (m, 1H), 2.79-2.74 (m, 1H), 2.68-2.52 (m, 1H), 2.48-2.27 (m, 9H), 2.18 (s, 3H), 2.02-1.90 (m, 1H), 1.76-1.40 (m, 13H), 1.25 (brs, 40H), 0.90-0.85 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 779.7, Measured m / z = 779.7.

[0234] Lipid Synthesis Example 60 (Synthesis of (2-(3-(azetidin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(azetidin-1-yl)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that azetidine was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46. 1H NMR (300 MHz, CDCl3) δ4.45 (t, J = 4.8 Hz, 1H), 4.35 (s, 2H), 3.91 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.59 (d, J = 11.7 Hz. 2H), 3.16 (t, Mass spectrometry (ESI) calculated values [M+H] + m / z = 723.2, Measured m / z = 723.2.

[0235] Lipid Synthesis Examples 61 to 65 The lipids in Table 5 below were synthesized by changing the (2-(3-oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) and 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 to the aldehydes and cyclic amines in Table 5 below.

[0236] Lipid Synthesis Example 66 (Synthesis of (2-(3-(dipropylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate)) The target (2-(3-(dipropylamino)propyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that dipropylamine was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46. 1H NMR (300 MHz, CDCl3) δ4.47 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 3.92 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.7 Hz. 2H),2.43-2.30 (m, 8H), 1.64-1.39 (m, 16H), 1.25 (brs, 40H), 0.90-0.85 (m, 18H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 766.7, Measured m / z = 766.7.

[0237] Lipid Synthesis Examples 67 to 70 The lipids in Table 6 below were synthesized by changing the 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 to a secondary amine in Table 6 below.

[0238] Lipid Synthesis Example 71 (Synthesis of (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate)) The target (2-(4-(dimethylamino)butyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate) was obtained in the same manner as in Lipid Synthesis Example 46, except that dimethylamine 2 M solution in THF was used instead of 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46, and (2-(4-oxobutyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-nonylundecanoate) was used instead of (2-(3-oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate). 1H NMR (300 MHz, CDCl3) δ4.45 (t, J = 4.8 Hz, 1H), 4.36 (s, 2H), 3.91 (d, J = 11.7 Hz, 2H), 3.83 (s, 2H), 3.60 (d, J = 11.7 Hz. 2H),2.37-2.25 (m, 10H), 1.68-1.40 (m, 14H), 1.25 (brs, 56H), 0.90-0.86 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 836.8, Measured m / z = 836.8.

[0239] Lipid Synthesis Examples 72 to 83 The lipids in Table 7 below were synthesized by replacing (2-(3-oxopropyl)-1,3-dioxane-5,5-diyl)bis(methylene) bis(2-hexyldecanoate) and 3-(methylamino)propan-1-ol in Lipid Synthesis Example 46 with the aldehydes and secondary amines in Table 7 below.

[0240] Lipid Synthesis Examples 84 to 90 Lipids in Table 8 below were synthesized by changing undecanoic acid in Lipid Synthesis Example 27-(1) and 2-nonylundecanoic acid in (2) to the carboxylic acids in Table 8 below.

[0241] Lipid Synthesis Example 91 (Synthesis of 3-(5,5-Bis(((2-hexyldecyl)oxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine) (1) 2,2-Bis(bromomethyl)propane-1,3-diol (3.93 mg), 2,2-dimethoxypropane (1.84 ml), and p-toluenesulfonic acid monohydrate (28.5 mg) were dissolved in acetone (15 ml) and stirred overnight at room temperature. Saturated aqueous sodium bicarbonate was added to the reaction solution, and the acetone was evaporated under reduced pressure. The residue was extracted three times with ethyl acetate, and the organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain 5,5-bis(bromomethyl)-2,2-dimethyl-1,3-dioxane (3.51 g). 1H NMR (300 MHz, CDCl3) δ 3.80 (s, 4H), 3.58 (s, 4H), 1.42 (s, 6H). (2) 5,5-Bis(bromomethyl)-2,2-dimethyl-1,3-dioxane (227 mg), 2-hexyldecan-1-ol (476 μL), and NaH (60%, dispersion in paraffin liquid) (66 mg) were suspended in DMF (2 mL) and stirred at 100 °C for 4 days. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated with sodium sulfate. After filtration, the solvent was evaporated under reduced pressure. The residue was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to obtain a crude product containing 5,5-bis(((2-hexyldecyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane. (3) Methanol (8 ml), DCM (2.0 ml), and HCl (4 mol / L in 1,4-dioxane, 1.0 ml) were added to the crude product containing 5,5-Bis(((2-hexyldecyl)oxy)methyl)-2,2-dimethyl-1,3-dioxane, and the mixture was stirred at room temperature for 4 hours. An excess amount of sodium bicarbonate was added to the reaction solution, and the mixture was concentrated under reduced pressure. Water and ethyl acetate were added to the residue, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then sodium sulfate was added for dehydration. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 2,2-bis(((2-hexyldecyl)oxy)methyl)propane-1,3-diol (76.1 mg). 1H NMR (300 MHz, CDCl3) δ 3.63 (d, J = 6.1 Hz, 4H), 3.49 (s, 4H), 3.30 (d, J = 5.6 Hz, 4H), 2.78 (t, J = 6.1 Hz, 2H), 1.58-1.51 (m, 2H), 1.26 (brs, 48H), 0.90-0.86 (m, 12H). (4) In a vessel equipped with a calcium chloride tube, 2,2-bis(((2-hexyldecyl)oxy)methyl)propane-1,3-diol (76.1 mg), 4,4-dimethoxy-N,N-dimethylbutan-1-amine (25.9 μL), p-toluenesulfonic acid monohydrate (29.7 mg), triethyl orthoformate (26.0 μL), and 2,2-bis(((2-hexyldecyl)oxy)methyl)propane-1,3-diol (76.1 mg) were added. Aqueous hexane (1.0 mL) and toluene (1.0 mL) were added and stirred overnight at room temperature. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine, and then sodium sulfate was added for dehydration. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to obtain the desired 3-(5,5-bis(((2-hexyldecyl)oxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine (62.6 mg). 1 H NMR (300 MHz, CDCl3) δ 4.45 (t, J = 4.2 Hz, 1H), 3.90 (d, J = 11.6 Hz, 2H), 3.61 (d, J = 11.6Hz, 2H), 3.57 (s, 2H), 3.29 (d, J = 5.9 Hz, 2H), 3.17 (d, J = 5.73 Hz, 2H), 3.12 (s, 2H), 2.28 (t, J = 7.0 Hz, 2H), 2.22 (s, 6H), 1.68-1.51 (m, 6H), 1.26 (brs, 48H), 0.90-0.86 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 682.7, Measured m / z = 682.7.

[0242] Lipid Synthesis Example 92 (Synthesis of 3-(5,5-bis((nonadecan-10-yloxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine) The target 3-(5,5-bis((nonadecan-10-yloxy)methyl)-1,3-dioxan-2-yl)-N,N-dimethylpropan-1-amine was obtained in the same manner as in Lipid Synthesis Example 86, except that nonadecan-10-ol was used instead of 2-hexyldecan-1-ol in Lipid Synthesis Example 91-(2). 1 H NMR (300 MHz, CDCl3) δ 4.47 (t, J = 4.7 Hz, 1H), 3.90 (d, J = 11.5 Hz, 2H), 3.64-3.59 (m, 4H), 3.25-3.21 (m, 1H), 3.14 (s, 2H), 3.10-3.07 (m, 1H), 2.29 (t, J = :6.6 Hz, 2H), 2.23 (s, 6H), 1.61-1.33 (m, 12H), 1.26 (brs, 56H), 0.89-0.87 (m, 12H). Mass spectrometry (ESI) calculated value [M+H] + m / z = 766.8, Measured m / z = 766.8.

[0243] Lipid Synthesis Example 93 (Synthesis of (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl) bis(2-nonylundecanoate)) (1) Dimethylmalonate (1.32 g), potassium carbonate (2.76 g), TBAB (322 mg), and (3-bromopropoxy)(tert-butyl)dimethylsilane (6.33 g) were added to toluene (30 ml) and stirred overnight at 85°C. Saturated aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain dimethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate (2.55 g). 1 H NMR (300 MHz, CDCl) δ 3.74 (s, 6H), 3.63 (t, J = 6.2 Hz, 2H), 3.42 (t, J = 7.6 Hz, 1H), 2.00-1.93 (m, 2H), 1.60-1.49 (m, 2H), 0.89 (s, 9H), 0.04 (s, 6H). (2) NaH (60%, dispersion in paraffin liquid) (600 mg) was suspended in THF (20 ml) and stirred for 5 min under ice cooling. Dimethyl 2-(3-((tert-butyldimethylsilyl)oxy)propyl)malonate (3.05 g) was added to the reaction mixture and stirred for an additional 5 min. (3-bromopropoxy)(tert-butyl)dimethylsilane (3.09 g) was added, and the mixture was stirred overnight at room temperature and then at 80°C for another day. A saturated aqueous solution of ammonium chloride was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain dimethyl 2,2-bis(3-((tert-butyldimethylsilyl)oxy)propyl)malonate (939 mg). 1H NMR (300 MHz, CDCl) δ 3.71 (s, 6H), 3.59 (t, J = 6.3 Hz, 4H), 1.96-1.90 (m, 4H), 1.42-1.35 (m, 4H), 0.88 (s, 18H), 0.03 (s, 12H). (3) Dimethyl 2,2-bis(3-((tert-butyldimethylsilyl)oxy)propyl)malonate (1.91 g) was dissolved in THF (10 mL) and stirred for 10 minutes under ice-cooling. To the reaction solution was added sodium bis(2-methoxyethoxy)aluminum hydride solution (≥60 wt. % in toluene) (4.0 mL), and the mixture was further stirred at room temperature overnight. The reaction was quenched by dropwise addition of aqueous sodium hydroxide solution under ice-cooling. The mixture was extracted three times with ethyl acetate, and the organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 2,2-bis(3-((tert-butyldimethylsilyl)oxy)propyl)propane-1,3-diol (607 mg). 1H NMR (300 MHz, CDCl) δ 3.60 (t, J = 6.1 Hz, 4H), 3.56 (d, J = 5.8 Hz, 4H), 2.61 (t, J = 5.8 Hz, 2H), 1.52-1.41 (m, 4H), 1.34-1.26 (m, 4H), 0.89 (s, 18H), 0.05 (s, 12H). (4) 2,2-Bis(3-((tert-butyldimethylsilyl)oxy)propyl)propane-1,3-diol (512 mg) and imidazole (293 mg) were dissolved in DCM (4 ml), and TBDPSCl (835 mg) was added under ice cooling. The mixture was stirred at room temperature for 3 days. Aqueous ammonium chloride solution was added to the reaction solution, and the mixture was extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 8,8-bis(((tert-butyldiphenylsilyl)oxy)methyl)-2,2,3,3,13,13,14,14-octamethyl-4,12-dioxa-3,13-disilapentadecane (660 mg). 1H NMR (300 MHz, CDCl) δ 7.64-7.62 (m, 8H), 7.42-7.28 (m, 12H), 3.51-3.46 (m, 8H), 1.28 (brs, 8H), 1.04 (s, 18H), 0.87 (s, 18H), 0.01 (s, 12H). (5) 8,8-Bis(((tert-butyldiphenylsilyl)oxy)methyl)-2,2,3,3,13,13,14,14-octamethyl-4,12-dioxa-3,13-disilapentadecane (539 mg) was dissolved in THF (12 ml), and ultrapure water (1.92 ml) and 5 M HCl-dioxane (480 μl) were added. The mixture was stirred for 30 min. Aqueous sodium bicarbonate solution was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 4,4-bis(((tert-butyldiphenylsilyl)oxy)methyl)heptane-1,7-diol (338 mg). 1H NMR (300 MHz, CDCl3) δ 7.66-7.62 (m, 8H), 7.44-7.31 (m, 12H), 3.52 (s, 4H), 3.46-3.41 (m, 4H), 1.28-1.18 (m, 8H), 1.06 (s, 18H). (6) 4,4-Bis(((tert-butyldiphenylsilyl)oxy)methyl)heptane-1,7-diol (134 mg), 2-nonylundecanoic acid (138 mg), and DMAP (2.44 mg) were dissolved in DCM (1 ml), and EDCI (99.7 mg) was added and the mixture was stirred overnight at room temperature. Aqueous ammonium chloride was added to the reaction solution, which was then extracted three times with DCM. The organic layer was washed with saturated brine and then dehydrated with sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography [eluent: hexane / ethyl acetate] to obtain 4,4-bis(((tert-butyldiphenylsilyl)oxy)methyl)heptane-1,7-diyl bis(2-nonylundecanoate) (229 mg). (7) THF (2 ml), acetic acid (343 μl), and TBAF (1.0 mol / L in THF, 4 ml) were added to 4,4-Bis(((tert-butyldiphenylsilyl)oxy)methyl)heptane-1,7-diyl bis(2-nonylundecanoate) (229 mg) and stirred at room temperature for 2 hours. Aqueous sodium bicarbonate was added to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by silica gel chromatography (eluent: hexane / ethyl acetate) to give 4,4-bis(hydroxymethyl)heptane-1,7-diyl bis(2-nonylundecanoate) (51.3 mg). 1H NMR (300 MHz, CDCl) δ 4.06 (t, J = 6.4 Hz, 4H), 3.58 (s, 4H), 2.35–2.26 (m, 2H), 1.62–1.30 (m, 16H), 1.25 (s, 56H), 0.90–0.86 (m, 12H). (8) 4,4-Bis(hydroxymethyl)heptane-1,7-diyl bis(2-nonylundecanoate) (34.1 mg) was added to 4,4-dimethoxy-N,N-dimethylbutan-1-amine (10.2 μL), p-toluenesulfonic acid monohydrate (10.7 mg), and toluene (10 ml) and stirred overnight at 85 °C. An excess amount of saturated aqueous sodium bicarbonate was added to the reaction solution, which was then extracted three times with ethyl acetate. The organic layer was washed with saturated brine and then dehydrated by adding sodium sulfate. After filtration, the solvent was distilled off under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography [eluent: DCM / methanol] to obtain the desired (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl)bis(2-nonylundecanoate). 1 H NMR (300 MHz, CDCl3) δ 4.44 (t, J = 3.8 Hz, 1H), 4.09 (t, J = 6.5 Hz, 2H), 4.02 (t, J = 6.3 Hz, 2H), 3.75 (d, J = 11.4 Hz, 2H), 3.37 (t, J = Mass spectrometry (ESI) calculated value [M+H] + m / z = 878.8, Measured m / z = 878.8.

[0244] Lipid Synthesis Example 94 (Synthesis of (2-(3-(Dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl)bis(2-hexyldecanoate)) The target (2-(3-(dimethylamino)propyl)-1,3-dioxane-5,5-diyl)bis(propane-3,1-diyl)bis(2-hexyldecanoate) was obtained in the same manner as in Lipid Synthesis Example 88, except that 2-hexyldecanoic acid was used instead of 2-nonylundecanoic acid in Lipid Synthesis Example 93. 1 H NMR (300 MHz, CDCl3) δ 4.44 (t, J = 4.1 Hz, 1H), 4.10 (t, J = 6.5 Hz, 2H), 4.02 (t, J = 6.3 Hz, 2H), 3.75 (d, J = 11.4 Hz, 2H), 3.38 (d, J = Mass spectrometry (ESI) calculated value [M+H] + m / z = 766.7, Measured m / z = 766.7.

[0245] II. Preparation and Evaluation of Lipid Nanoparticles <Buffer Preparation> Citrate buffer: 6.90 g of citric acid (Fujifilm Wako, product code: 030-05525) and 4.15 g of sodium citrate dihydrate (Aldrich, catalog number: W302600) were added to ultrapure water to prepare 1 L of citrate buffer (pH 3.5). HEPES buffer: 90.0 g of sucrose (Fujifilm Wako, product code: 196-00015) and 20 ml of HEPES buffer (1 mol / L, Nacalai Tesque, product code: 17557-94) were added to ultrapure water to prepare 1 L of HEPES buffer (pH 7.45). TE buffer (20X), RNase-free (Invitrogen™, product code: T11493) was diluted 20-fold with DEPC-Treated Water (Nippon Gene, product code: 314-90205) to prepare TE buffer (pH 7.5). PBS(-) Dulbecco's Phosphate-Buffered Saline (Nacalai Tesque, product code: 14249-24) was used.

[0246] <Preparation of Lipid Nanoparticles> Lipid nanoparticles were prepared by the alcohol dilution method using a microfluidic device. The microfluidic device used was the NanoAssemblr® Benchtop (PRECISION NANOSYSTEMS). First, a pH-sensitive cationic lipid, DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine), cholesterol, and PEG-DMG 2000 (NOF, SUNBRIGHT GM-020) were mixed in a molar ratio of 50:10:38.5:1.5 to prepare an ethanol solution with a total lipid concentration of 8 mM. Nucleic acid solutions were prepared by diluting CleanCap FLuc mRNA (TriLink BioTechnologies, product number L-7202-5) to 44.1 μg / ml and siRNA to 128 μg / ml with citrate buffer. 0.650 ml of the prepared lipid solution and 1.95 ml of the nucleic acid solution were mixed in a microfluidic device at a total flow rate of 12 ml / min, and 2.20 ml of the mixed solution was collected in a tube containing 2.00 ml of HEPES buffer. The collected lipid nanoparticle solution was placed in a dialysis membrane with a molecular weight cutoff of 12-14 kD (REPLIGEN Spectra / Por® 4) and dialyzed overnight at 5°C using HEPES buffer as the dialysate. The dialyzed lipid nanoparticle solution was concentrated using an ultrafiltration unit (Amicon® Ultra-15), and the lipid nanoparticle solution was collected. The collected lipid nanoparticle solution was diluted with HEPES buffer to a nucleic acid concentration of 0.01 mg / ml and stored at -80°C until use.

[0247] <Evaluation of Encapsulation Rate of Lipid Nanoparticles> The nucleic acid encapsulation rate in lipid nanoparticles was measured using Ribogreen reagent (Quant-it™, product number R114491). Measurement solution A was prepared by diluting the lipid nanoparticle solution with TE buffer to a nucleic acid concentration of approximately 8 μg / ml. Measurement solution B was also prepared by diluting the lipid nanoparticle solution with TE buffer to a nucleic acid concentration of approximately 1.2 μg / ml and 1% (w / w) X-triton 100 (Sigma-Aldrich, T8787-50ML). 100 μl of the measurement solution and 100 μl of Ribogreen reagent were mixed on a 96-well microplate and incubated at room temperature for 5 minutes. The fluorescence intensity was measured at an excitation wavelength of 485 nm and a measurement wavelength of 528 nm. The nucleic acid concentration was calculated using a calibration curve prepared over a nucleic acid concentration range of 0 to 2.5 μg / ml. The encapsulation rate of lipid nanoparticles was calculated using the following formula: Encapsulation rate % = (nucleic acid concentration of measurement solution B (μg / ml) - nucleic acid concentration of measurement solution A (μg / ml)) ÷ nucleic acid concentration of measurement solution B (μg / ml) × 100

[0248] <Measurement of the average particle size of lipid nanoparticles> The lipid nanoparticle solution was diluted 25 times with PBS(-) to prepare a measurement solution. The average particle size (z) and polydispersity index (PDI) of the nanoparticles in the measurement solution were measured using an analyzer "Zetasizer Nano ZSP" (Malvern).

[0249] <In vitro mRNA delivery into cultured cells> 24 hours before transfection, HepG2 or CHO-K1 cells were seeded at 5,000 cells / well in a 96-well plate (Corning, #3904). The media used were DMEM, low glucose, pyruvate (Gibco, #11885-084) or DMEM / F-12 (Gibco, #11320-033) adjusted to a final concentration of 10% FBS and 1% penicillin / streptomycin, respectively. FLuc mRNA (TriLink Bio Technologies, L-7202-5)-encapsulated LNPs were transfected at 1 ng / well and incubated at 37°C in a 5% CO2 atmosphere. 24 hours after transfection, luciferase expression was quantified using the ONE-Glo™ + Tox Luciferase Reporter and Cell Viability Assay (Promega, E7120) to determine the drug transduction index. The assay conditions followed the manufacturer's protocol. GF-AFC was added and incubated at 37°C and 5% CO2 for 2 hours, after which the fluorescence intensity was measured to estimate the relative number of viable cells. Next, the substrate for the ONE-Glo™ Luciferase Assay was added, and luminescence intensity was measured 3 minutes later. The luminescence intensity was corrected for the number of viable cells to determine the drug transduction index. The drug transduction index was calculated by multiplying the luminescence intensity corrected for the number of viable cells by 10. 5 The drug transduction index is shown in Table 9. A larger drug transduction index indicates that a larger amount of mRNA (drug) was transduced into the cells.

[0250] The nucleic acid encapsulation rate, mean particle size, polydispersity index (PDI), and in vitro drug uptake index of lipid nanoparticles using each lipid from pH-sensitive cationic lipid Synthesis Examples 1 to 29 are summarized in Table 9-1 below. The nucleic acid encapsulation rate, mean particle size, and polydispersity index (PDI) of lipid nanoparticles using each lipid from pH-sensitive cationic lipid Synthesis Examples 30 to 89 are also summarized in Tables 9-2 and 9-3 below. The "HEPES buffer" in Table 9-1 refers to an experiment in which HEPES buffer containing no lipid nanoparticles was used instead of the lipid nanoparticle-containing solution suspended in HEPES buffer used in the Synthesis Examples. Lipid nanoparticles using each lipid from pH-sensitive cationic lipid Synthesis Examples 1 to 29 were confirmed to have a high mRNA encapsulation rate and also showed an increased drug uptake index. It is clear that lipid nanoparticles using each lipid from pH-sensitive cationic lipid Synthesis Examples 1 to 29 were able to deliver the encapsulated mRNA into cells. It was confirmed that the lipid nanoparticles using each of the lipids in pH-sensitive cationic lipid synthesis examples 30 to 89 had a high mRNA encapsulation rate.

[0251]

[0252]

[0253] <Introduction of siRNA into in vitro cultured cells> HepG2 cells were seeded at 2,500 cells / well in a 96-well plate (Corning, #3904) 24 hours before transfection. The medium used was DMEM, low glucose, pyruvate (Gibco, #11885-084) adjusted to a final concentration of 10% FBS and 1% penicillin / streptomycin. LNPs containing siRNA against human PLK1 (polo-like kinase) or luciferase siRNA were added to the culture medium to a final concentration of 1 nM, followed by transfection and incubation at 37°C in a 5% CO2 environment. PLK1 is a kinase responsible for progression through the M phase of the cell cycle, and its gene knockdown is known to suppress cell proliferation. In this evaluation, the degree of gene knockdown was assessed using the percentage of viable cells as an indicator. 72 hours after transfection, the percentage of viable cells was measured using the CellTiter-Fluor™ Cell Viability Assay (Promega, G6080). Assay conditions followed the manufacturer's protocol. GF-AFC was added and incubated at 37°C in a 5% CO2 environment for 2 hours. Fluorescence intensity was then measured to determine the number of viable cells. The viability index, which is the ratio of the number of viable cells treated with siRNA against human PLK1 (siPLK1) to the number of viable cells treated with siRNA against luciferase (siLuc), was calculated using the following formula: Calculation formula: Viable cell index (%) = Number of viable cells treated with siPLK1 / Number of viable cells treated with siLuc × 100. The lipid compositions of the siPLK1-containing lipid nanoparticles and the siLuc-containing lipid nanoparticles were kept the same, and the number of viable cells treated with siLuc was used to correct for the effect of siRNA-containing LNPs coming into contact with cells. When siPLK1 is introduced into cells, cell proliferation is inhibited. Therefore, if the siRNA delivery capacity of siPLK1-containing lipid nanoparticles is high, the viable cell index will be low.Therefore, a lower viability index (%) indicates a higher efficiency of siPLK1 transfection into cells. Comparative Example 1 was performed in the same manner, except that instead of adding lipid nanoparticles using pH-sensitive cationic lipids, siPLK1 was transfected according to the Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, product number 13778-150) protocol. Comparative Example 2 was performed in the same manner as for lipid nanoparticles using pH-sensitive cationic lipids, except that siRNA was added to the culture medium at a final concentration of 1 nM without the use of lipid nanoparticles.

[0254] The nucleic acid encapsulation rate, average particle size, polydispersity index (PDI), and viability index of lipid nanoparticles using each of the lipids in pH-sensitive cationic lipid Synthesis Examples 1, 4, 10, 14, and 15 are shown in Table 10 below. The sequences of the encapsulated siRNAs are also shown in Table 11. It was confirmed that lipid nanoparticles using the pH-sensitive cationic lipids of the present invention have a high siRNA encapsulation rate. Furthermore, since all of the lipid nanoparticles using the pH-sensitive cationic lipids of the present invention have a low viability index, it was revealed that the pH-sensitive cationic lipids of the present invention are useful for introducing siRNA into cells.

[0255]

[0256]

[0257] From the above results, lipid nanoparticles using the pH-sensitive cationic lipid of the present invention have a high mRNA encapsulation rate and an increased drug introduction index, which allows the encapsulated mRNA to be efficiently introduced into cells.Furthermore, the pH-sensitive cationic lipid of the present invention is useful for introducing siRNA into cells.

[0258] Evaluation of mRNA Delivery and Expression in Experimental Animals: Balb / c mice (n=3) aged 6 to 7 weeks were intravenously administered the lipid nanoparticle solution at a dose of 0.1 mg kg-1 of mRNA encapsulated therein. Six hours after administration, 1.5 mg of VivoGlo Luciferin In Vivo Grade (Promega, US) dissolved in PBS(-) was injected into the tail vein, and the mice were sacrificed by exsanguination 3 minutes later. The lungs, liver, spleen, pancreas, and kidneys were collected from the mice, and luciferin luminescence was imaged using an IVIS Imaging System. Avg. Radiance [p / s / cm2 / sr] was calculated by specifying the region of interest for each organ using the ROI tool. The mRNA expression levels in the liver and spleen of lipid nanoparticles using each of the pH-sensitive cationic lipid synthesis examples 1 to 89 are summarized in Table 12. For comparison, the results for HEPES administration are also shown in Table 12. All of the pH-sensitive cationic lipids of the present invention were confirmed to emit luminescence in the liver or spleen that was greater than that observed with HEPES administration, demonstrating their ability to deliver mRNA in experimental animals.

[0259]

Claims

1. General formula I: General formula II: Or, general formula III: wherein L is independently H, straight-chain C １－１０ Alkyl or branched C ３－１０ Alkyl, or and A １ is C １－６ alkylene or phenylene; ２ is —OC(═O)—, —OC(═O)—O—, or —OC(═O)—NH—, or is absent; ３ is C １－６ alkylene or absent, A ４ is a 4- to 15-membered nitrogen-containing heterocyclyl, wherein the nitrogen-containing heterocyclyl is unsubstituted or １－４ substituted with at least one selected from the group consisting of alkyl, hydroxy, and oxo; or A ４ teeth, where B １ and B ２ are each independently １－４ Alkyl, hydroxy C １－４ Alkyl, C １－４ Alkoxy C １－４ alkyl, or 4- to 6-membered heterocyclyl, wherein said 4- to 6-membered heterocyclyl is unsubstituted or １－４ is substituted with at least one selected from the group consisting of alkyl, hydroxy, and oxo; １ and M ２ are independently —O—, —O—CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -CH ２ -CH ２ -, -O-C(O)-C-C-C(O)-, -O-C(O)-CH ２ -CH ２ -C(O)-O-, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -, -OC(O)-CH ２ -CH ２ -C(O)-NH-, -O-CH ２ -CH ２ -CH ２ -O-C(O)-, -C-C(O)-O-, -CH ２ -C(O)-O-CH ２ -, -C-CH ２ -O-C(O)-, -S-S-CH ２ - or -S-CH ２ -C-C-S-S-CH ２ - and R １ and R ２ are independently linear C １－３０ alkyl, branched C ３－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, sterol groups, and in general formulas I and III, at least one L is a compound represented by the formula (I), a salt thereof or a stereoisomer thereof.

2. The compound has general formula I, wherein one L is H and the other L is:

2. The compound of claim 1, a salt thereof or a stereoisomer thereof, wherein:

3. One L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ The compound of claim 2, a salt thereof, or a stereoisomer thereof, wherein:

4. One L is H and the other L is (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ The compound of claim 3, or a salt or stereoisomer thereof, wherein:

5. M １ and M ２ The compound, salt or stereoisomer thereof according to claim 4, wherein:

6. M １ and M ２ is -O-, -O-CH ２ -, -O-C(O)-, -O-C(O)-O-, -O-C(O)-O-CH ２ -, -OC(O)-CH ２ -, -OC(O)-CH ２ -CH ２ -, -OC(O)-CH ２ -CH ２ -C(O)-O-CH ２ -or-CH ２ -CH ２ The compound, salt or stereoisomer thereof according to claim 5, wherein the compound is —O—C(O)—.

7. R １ and R ２ are independently linear C １－３０ Alkyl, straight chain C ２－３０ Alkenyl, branched C ３－３０ Alkenyl, adamantyl, and R ４ and R ５ are independently linear C １―１６ The compound of claim 6, or a salt or stereoisomer thereof, wherein:

8. One L is H and the other L is and R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl, provided that R ａ and R ｂ The total number of carbon atoms in R is 4 or less, ｃ is a straight chain C ２－６ The compound of claim 7, a salt thereof, or a stereoisomer thereof, wherein: R is alkylene.

9. R １ and R ２ is independently and R ４ and R ５ are independently linear C １―１１ alkyl, where R ４ and R ５ The compound, salt or stereoisomer thereof according to claim 7, wherein the total number of carbon atoms is 10 to 22.

10. R １ and R ２ is independently and R ４ and R ５ are independently linear C ５―１１ alkyl, where R ４ and R ５ The compound, salt or stereoisomer thereof according to claim 7, wherein the difference in the number of carbon atoms between 11. The compound is represented by general formula II, wherein L is a linear C １－５ alkyl, M １ and M ２ is -O-C(O)-, and R １ and R ２ are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６ The compound of claim 1 , a salt thereof, or a stereoisomer thereof, wherein:

12. The compound has general formula III, wherein one L is H and the other L is: (In the formula, R ａ and R ｂ are independently methyl, ethyl, n-propyl, or isopropyl; R ｃ is a straight chain C １－６ alkylene), and M １ and M ２ is -O-C(O)-, and R １ and R ２ are the same and are linear C １－３０ Alkyl, straight chain C ２－３０ alkenyl, or and R ４ and R ５ are independently linear C １―１６ The compound of claim 1 , a salt thereof, or a stereoisomer thereof, wherein:

13. The compound of claim 1, its salt or stereoisomer, wherein the compound is represented by any of the following:

14. Lipid nanoparticles containing the compound of claim 1, its salt or stereoisomer.

15. The lipid nanoparticle of claim 14, further comprising a sterol and a polyalkylene glycol-modified lipid.

16. The lipid nanoparticle of claim 14, which contains a nucleic acid.

17. The lipid nanoparticle of claim 16, wherein the nucleic acid is mRNA or siRNA.

18. A pharmaceutical composition comprising the lipid nanoparticles of claim 14.

19. The pharmaceutical composition according to claim 18, which is used in gene therapy.

20. A method for expressing a foreign gene, comprising administering the lipid nanoparticles described in claim 14, which encapsulate a foreign gene to be expressed in a cell, to a subject animal (excluding humans), and expressing the foreign gene in the cells of the subject animal.

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